Immunogenic cd19 peptides

ABSTRACT

The invention provides relatively short immunogenic peptides derived from CD19 antigens, and biologically active variants thereof, which elicit an immune response. Nucleic acids encoding the immunogenic peptides and antibodies specific for the peptides are also provided. The immunogenic peptides can be included in pharmaceutical compositions, such as cancer vaccines, and used for the treatment of cancer.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/884,862, filed Jul. 2, 2004, which claims priority to U.S.Provisional Application No. 60/484,689, filed Jul. 3, 2003, the entiredisclosure of which are incorporated by reference and for all purposesas if fully set forth herein.

FIELD OF INVENTION

The present invention relates to immunogenic peptides. More particularlythe present invention relates to CD19 or CD20 peptides that elicit animmune response.

BACKGROUND OF THE INVENTION

B-cell malignancies comprise a heterogeneous group of neoplasmsincluding acute lymphocytic leukemia, chronic lymphocytic leukemia, andB-cell lymphomas. An estimated 65,000 new cases are diagnosed annuallyin the United States. Current therapeutic strategies can be effectivebut the majority of patients ultimately relapse and die of theirdisease. One promising approach for targeting B-cell malignanciesinvolves the cellular immune system through activation of highlyefficient T-lymphocytes that mediate key functions such as cytotoxicity,cytokine production, regulation of effector cells, and induction ofimmunological memory. Earlier clinical studies have shown that dendriticcell vaccination for the priming of naïve T cells can generatetumor-specific CTLs and induce remission in pre-treated patients withhuman B-cell malignancies. In addition, infusion of HLA-matchedallogeneic T lymphocytes has been shown to induce durable long-termremissions in relapsed lymphomas, chronic B-lymphocytic leukemia (CLL),multiple myeloma, or EBV-associated lymphoproliferative disease afterstem cell transplantation. However, only a limited percentage ofpatients with B-cell malignancies will achieve complete remissionfollowing donor lymphocyte infusion and the patients are at risk ofdeveloping graft-versus-host-disease, which can be associated withsignificant morbidity and mortality. Therefore, developing peptide-basedimmunotherapies against specific over-expressed tumor-associatedantigens offer an attractive approach for boosting patients' immunesystem to treat recurrent B-cell malignancies.

CD19, a 95 kDa B lineage-specific transmembrane glycoprotein, functionsas a central response regulator in B cells and offers many uniquecharacteristics that make it a relevant target for developingimmunotherapeutic strategies. With the important exception ofhematopoietic stem cells, CD19 is expressed during all stages of on Bcell differentiation, is down regulated on plasma cells,¹⁵ and ismaintained on cells that have undergone neoplasic transformation. It isexpressed on >95% of cells in patients with B cell lymphoma, chronicB-lymphocytic leukemia, and on the acute B-lymphocytic leukemiaprogenitor cells. The CD19 antigen is also internalized after binding toantibody. Studies have also shown that CD19 expression is maintaineddespite loss of CD20 expression following treatment with anti-CD20antibodies.

CD20 is a non-glycosylated 33-37 kDa integral membrane phosphoproteininvolved in regulation of B-cell proliferation and differentiation. Itis expressed slightly later in B-cell development than CD19, is notrapidly internalized, is expressed at a high surface density on the vastmajority of lymphomas, and is eventually down-regulated on terminallydifferentiated plasma cells. Recently, in the treatment of thesecancers, clinical work has focused on passive therapy using rituximab, amonoclonal antibody directed against the CD20 antigen, either alone orcoupled to a radioactive compound. Although favorable clinical responseshave been observed, these antibodies alone are not curative with mostresponders achieving only partial remissions with a mean time to diseaseprogression of 13.2 months following antibody treatment.

It is appealing to identify, at a molecular level, the antigens that maybe effective immunogens for development of protective immunity againstcancer cells. Identifying these antigens would allow vaccination withimmunogenic cancer-associated molecules rather than with anuncharacterized mixture of tumor molecules including both immunogenicand immunosuppressive components. Because T cells are critical for theeradication of tumors, it is necessary to understand the nature of theantigens recognized by these cells. CD19 and CD20 are well known as theB cell lymphoma associated surface antigen due to the over-expression onthose cells. Monoclonal antibodies to CD20 such as rituximab have beensuggested as an effective immunotherapy for B cell lymphomas. However,as set forth above, treatment with rituximab does not result in a cure.Vaccination is an alternative immunotherapeutic approach for thetreatment of lymphoma. Peptide vaccines have been the subject ofpre-clinical and clinical studies for the treatment of various types oftumors, including melanoma, leukemia, and breast cancer.

There continues to be a strong need for methods of diagnosing and viabletreatment regimens for diseases or conditions, such as B cell lymphomas,associated with the expression of CD19 and/or CD20.

SUMMARY OF THE INVENTION

One aspect of the present invention provides isolated or recombinantleukemic antigens comprising a fragment of CD19 or CD20 antigen or avariant thereof that is capable of stimulating a cytotoxic T-lymphocytereaction. The fragment or variant thereof can be 8, 9, 10, 11 or 12amino acids in length. In some embodiments, the fragment or variantthereof can be up to 50 or 80 amino acids in length. In someembodiments, when the fragment is a CD20 fragment, the fragment is not a44 amino acid extracellular domain of CD20.

In some embodiments, the fragments have defined anchor positions. Insome embodiments, the second position of the fragment is L or I. Inthese and other embodiments, the ninth position, relative to the otheranchor fragments, is L or V. In these and other embodiments, the firstanchor position is preferably not N, E or P. In these and otherembodiments, the third anchor position, relative to the first anchorposition, is preferably not N or E. In these and other embodiments, thefourth anchor position, relative to the first anchor position, ispreferably not R, K, H, or A. In these and other embodiments, the fifthanchor position, relative to the first anchor position, is preferablynot P. In these and other embodiments, the seventh anchor position,relative to the first anchor position, is preferably not R, K or H. Inthese and other embodiments, the eighth anchor position, relative to thefirst anchor position, is preferably not D, E, R, K, or H. In these andother embodiments, the ninth anchor position, relative to the firstanchor position, is preferably not R, K or H.

In some aspects the isolated leukemic antigen is immunologicallyrecognized by MHC restricted T-lymphocytes that are HLA-A2.1 restricted.When the fragment is from CD19 or a variant thereof the identifiedleukemic antigen can include the amino acid sequence RLLFFLLFL (SEQ IDNO: 1), TLAYLIFCL (SEQ ID NO: 2), LLFLTPMEV (SEQ ID NO: 3), KLMSPKLYV(SEQ ID NO: 4), or LLFFLLFLV (SEQ ID NO: 5). When the fragment is fromCD20 or a variant thereof the identified leukemic antigen can includethe amino acid sequence SLFLGILSV (SEQ ID NO: 6), AISGMILSI (SEQ ID NO:7), FIRAHTPYI (SEQ ID NO: 8), SLNFIRAHT (SEQ ID NO: 9), LKMESLNFI (SEQID NO: 10), SHFLKMESL (SEQ ID NO: 11), or YLFLGILSV (SEQ ID NO: 12).Suitable fragments can also include these sequences with or without oneor more, such as one, two, three, four, five or more conservative ornonconservative amino acid substitutions. The isolated leukemic antigencan also be combined with one or more co-immunostimulatory molecules.

The present invention also provides a method for stimulating an immuneeffector cell response achieved by contacting the isolated leukemicantigen with an immune effector cell which stimulates the immuneeffector cell to respond against the isolated leukemic antigen. In somemethods the immune effector cell is a naïve T-lymphocyte or a memoryT-lymphocyte. The method can be performed by contacting the isolatedleukemic antigen with an antigen presenting cell, in vivo or in vitro,such that the antigen presenting cell contacts the isolated leukemicantigen with the immune effector cell. Suitable antigen presenting cellsare dendritic cells or T2 cells.

The present invention also pertains to immune effector cells and antigenpresenting cells produced by these methods. Nucleic acids encoding thepresent isolated leukemic antigens also form part of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a timeline of events performed for the generation of CD20peptide-specific cytotoxic T-lymphocytes;

FIGS. 2 a and 2 b show the cytotoxic activity of CD19 and CD20peptide-specific CTLs against ST486 (Burkett's lymphoma cell line);

FIG. 3 shows the cytotoxic activity of CD20 peptide-specific CTLs toIFN-γ/TNF-α treated ST486 cells;

FIG. 4 shows the expansion of CD20 peptide specific cytotoxicT-lymphocytes over time using CD3/CD28 beads and IL-2; and

FIG. 5 shows the cytotoxic activity of expanded CD20 peptide specificcytotoxic T-lymphocytes to ST486 at different effector:target cellratios.

FIG. 6 shows the IFN-γ released by CD20 peptide specific cytotoxicT-lymphocytes to ST486 compared against unstimulated cytotoxicT-lymphocytes.

DETAILED DESCRIPTION

The present invention encompasses fragments of the CD19 or CD20 antigen,variants, isoforms and other mammalian homologs thereof which areimmunologically recognized by T lymphocytes of the immune system. CD19antigen is a member of the Ig superfamily and is generally expressed bynormal B cells including early B cells. Gene structure is highlyconserved between the mouse and human homologs of this gene. Zhou etal., Immunogenetics, 35(2):102-111 (1992). CD19 is also found onfollicular dendritic cells, early cells of myelomonocytic lineage andmost stabilized B cells. The CD19 antigen is not present on T cells oron normal granulocytes.

The present invention further encompasses the antigen cancer epitope(s)which are contained in the tumor antigen. The antigenic cancer epitopespecifically causes a cellular mediated immune response by interactionwith T cells of the immune system. This interaction between theantigenic cancer epitope and the T cells causes the T cells to respondagainst, and prevent, eliminate or reduce the cancer in a mammal,including humans. The CD19 and CD20 peptides, nucleic acid moleculeswhich code for such peptides, CD19 and CD20 binding agents such asantibodies, antigen presenting cells (APCs) and/or immune system cells(e.g., T cells), antibodies against such peptides and nucleic acids, areuseful, inter alia, in diagnostic and therapeutic contexts. Thus, thepresent invention provides a cancer vaccine.

The CD19 antigen is discussed in Stamenkovic et al., J Exp Med,168:1205-1210 (1988); and Tedder et al., Immunol Today, 15:437 (1994).The CD19 antigen and its murine homolog are discussed in Tedder et al.,J Immunol, 143:712-717 (1989). The CD19 homolog of mus musculus isdiscussed in Otero et al., J Immunol, 170(1):73-83 (2003). The structureand domain organization of the CD19 antigen of human, mouse, and guineapig B lymphocytes is discussed by Zhou et al., J Immunol,147(4):1424-1432 (1991). The canine CD19 gene is discussed by Liu etal., Anim Genet. 29(1):64-65 (1998). The sequence of the CD19 homolog ofSus scrofa has been described by Sun et al., and is available athttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=protein&list_uids=18496846&dopt=GenPept.

The sequence for the CD19 antigen as reported in Tedder et al. anddisclosed in the SwissProt annotated protein record P15391 is asfollows:

(SEQ ID NO: 13)   1 MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLKGTSDGPTQQL TWSRESPLKP  61 FLKLSLGLPG LGIHMRPLAS WLFIFNVSQQ MGGFYLCQPGPPSEKAWQPG WTVNVEGSGE 121 LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWAKDRPEIWEGE PPCVPPRDSL 181 NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPKGPKSLLSLEL KDDRPARDMW 241 VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVLWHWLLRTGGW KVSAVTLAYL 301 IFCLCSLVGI LHLQRALVLR RKRKRMTDPT RRFFKVTPPPGSGPQNQYGN VLSLPTPTSG 361 LGRAQRWAAG LGGTAPSYGN PSSDVQADGA LGSRSPPGVGPEEEEGEGYE EPDSEEDSEF 421 YENDSNLGQD QLSQDGSGYE NFEDEPLGPE DEDSFSNAESYENEDEELTQ PVARTMDFLS 481 PHGSAWDPSR EATSLGSQSY EDMRGILYAA PQLHSIRGQPGPNHEEDADS YENNDNPDGP 541 DPAWGGGGRM GTWSTR.

The extracellular domain of the CD19 antigen comprises residues 20-291.Other domains or regions of CD19 are set forth in the following chart.Generally, a description of “potential” in the chart can be understoodas indicating that the underlying classification of the listed stretchof amino acids has not been completely elucidated.

Key Begin End Length Description SIGNAL 1 19 19 POTENTIAL CHAIN 20 556537 B-LYMPHOCYTE ANTIGEN CD 19 DOMAIN 20 291 272 EXTRACELLULAR(POTENTIAL) TRANSMEM 292 313 22 POTENTIAL DOMAIN 314 556 243 CYTOPLASMIC(POTENTIAL) DOMAIN 31 104 74 IG-LIKE C2-TYPE DOMAIN 1 DOMAIN 193 268 76IG-LIKE C2-TYPE DOMAIN 2 DISULFIDE 38 97 60 POTENTIAL DISULFIDE 200 26162 POTENTIAL CARBOHYD 86 86 1 N-LINKED (GLCNAX (POTENTIAL) CARBOHYD 125125 1 N-LINKED (GLCNAC . . . ) (POTENTIAL) CARBOHYD 138 138 1 N-LINKED(GLCNAC . . . ) (POTENTIAL) CARBOHYD 181 181 1 N-LINKED (GLCNAC . . . )(POTENTIAL) CARBOHYD 265 265 1 N-LINKED (GLCNAC . . . ) (POTENTIAL)

References discussing antibodies against the CD19 antigen include Bejceket al., Cancer Res, 55(11):2346-2351 (1995); and Tulpule et al., ProcAnn Meet Am Soc Clin Oncol, 13:A10 (1994). Rituximab is a monoclonalantibody that recognizes the CD20 antigen.

The CD20 antigen is a non-glycosylated phosphoprotein of approximately35 kD. The CD20 antigen is expressed on B lymphocytes synchronously withthe expression of surface IgM. Dörken et al., “Leucocyte Typing IV:White Cell Differentiation Antigens,” In: Knapp W., Dörken B, Gilks W Ret al., eds. Leucocyte Typing IV: White Cell Differentiation Antigens.Oxford: Oxford University Press, 46-48 (1989); Loken et al., Blood,70:1316-1324 (1987). The antigen is present on both resting andactivated B lymphocytes but is lost prior to differentiation into plasmacells. The CD20 antigen is found in both mantle-zone and germinal centerareas of secondary follicles of lymphoid tissue and may be expressed onfollicular dendritic cells (FDC) in germinal centers. Low-levelexpression of the CD20 antigen has been detected on a subpopulation of Tlymphocytes. Hultin et al., Cytometry, 14:196-204 (1993); Algino et al.,Am J Clin Pathol, 106(1):78-81 (July 1996). CD20 antigen expressiongenerally occurs after CD19 and CD10 expression but before CD21/22expression and surface immunoglobulin expression. CD20 expression may beassociated with acute leukemias, chronic lymphocytic leukemias andlymphomas, including B cell lymphomas, pre B ALL/LBL, lymphocytepredominant Hodgkin's lymphoma, spindle cell thymomas, and non-Hodgkin'slymphoma including mantle cell lymphoma. Scheuermann et al., LeukLymphoma, 18(5-6):385-397 (1995). Generally, CD20 is dimly expressed inboth benign and neoplastic T cells. Stem cells (B cell progenitors) inbone marrow lack the CD19 and CD20 antigens, allowing healthy B cells toregenerate after treatment and return to normal levels.

The CD20 antigen is discussed by Stamenkovic et al., J Exp Med,167(6):1975-1980 (1988); Tedder et al., Proc Natl Acad Sci USA,85(1):208-212 (1988); Tedder et al., J Immunol, 142 (7):2560-2568(1989); Einfeld et al., EMBO J, 7(3):711-717 (1988); and Strausberg etal., Proc Natl Acad Sci USA, 99(26):16899-16903 (2002). A mammalian CD20homolog is discussed by Macardle et al., J Bio. Regul Homeost Agents,16(2):136-138 (2002) (mus musculus). The CD20 homolog of Rattusnorvegicus is also known and the sequence is available athttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=protein&list_uids=27681331&dopt=GenPept.

The sequence for the CD20 antigen disclosed in the SwissProt annotatedprotein record P11836 is as follows:

(SEQ ID NO: 14)   1 MTTPRNSVNG TFPAEPMKGP IAMQSGPKPL FRRMSSLVGPTQSFFMRESK TLGAVQIMNG  61 LFHIALGGLL MIPAGIYAPI CVTVWYPLWG GIMYIISGSLLAATEKNSRK CLVKGKMIMN 121 SLSLFAAISG MILSIMDILN IKISHFLKME SLNFIRAHTPYINIYNCEPA NPSEKNSPST 181 QYCYSIQSLF LGILSVNLIF AFFQELVIAG IVENEWKRTCSRPKSNIVLL SAEEKKEQTI 241 EIKEEVVGLT ETSSQPKNEE DIEIIPIQEE EEEETETNFPEPPQDQESSP IENDSSP

The extracellular portion of CD20 comprises residues 142-188. Otherdomains or regions of CD20 are set forth in the following chart.Generally, a description of “potential” in the chart can be understoodas indicating that the underlying classification of the listed stretchof amino acids has not been completely elucidated.

Key Begin End Length Description DOMAIN 1 63 63 CYTOPLASMIC (POTENTIAL)TRANSMEM 64 84 21 POTENTIAL TRANSMEM 85 105 21 POTENTIAL TRANSMEM 121141 21 POTENTIAL EXTRACELL 142 188 46 POTENTIAL TRANSMEM 189 209 21POTENTIAL DOMAIN 210 297 88 CYTOPLASMIC (POTENTIAL) DISULFIDE 111 220110 PROBABLE

I. Proteins/Polypeptides/Peptides/Fragments Thereof

The compounds of this invention generally comprise a polypeptide,sometimes in isolated form, that stimulates a Th1 or CTL (cytotoxicT-lymphocyte) immune response in peripheral blood mononuclear cells(PBMCs). In particular, polypeptides 8-12 amino acids in lengthcomprising a stimulatory portion of the CD19 or CD20 antigen aredisclosed, such as a cancer or tumor rejection antigen. The peptides ofthe present invention can be from 8 to 80 amino acids in length, such as8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, 25, 30, 32, 35, 40,45, 50, 55, 60, 65, 70, 75 or 80 residues, or consecutive amino acids ofCD19 or CD20, in length. Preferably, when the peptide is longer inlength, the sequence of the peptide will substantially correspond withthe sequence of CD19 or CD20, for example within about 1, 2, 5, 10 or 20percent homology or identity. A cancer or tumor rejection antigen is anexample of a unique fragment of a polypeptide specific to cancer thatretains the functional capability of HLA binding and interaction withcytotoxic T lymphocytes. Tumor rejection antigens presented by HLAmolecules typically are 9 amino acids in length, although peptides of 8,9, 10, 11 and 12 and more amino acids, up to about 80, and can retainthe capability to interact with HLA and cytotoxic T lymphocytes to anextent effective to provoke a cytotoxic T lymphocyte response (see,e.g., Van den Eynde & Brichard, Curr Opin Immunol, 7:674-681 (1995);Coulie et al., Stem Cells, 13:393-403 (1995); and discussed in U.S. Pat.No. 6,271,019). In some embodiments, the polypeptides may encompassamino acid chains of any length, including full length proteins andportions thereof, wherein amino acid residues are linked by covalentpeptide bonds. Although CD19 and CD20 fragments are described herein forexemplary purposes, portions thereof, variants of the polypeptide (orportions thereof) and homologous proteins in other non-human mammals canalso be used. In one preferred embodiment, the polypeptides aresubstantially free of contaminating endogenous materials. In someembodiments, the peptides are derived from the signaling domain,extracellular domain, transmembrane spanning domain or cytoplasmic tailof CD19 or CD20. In some embodiments, the peptide is not a 44 amino acidextracellular domain of CD20 or peptide disclosed by Robert et al.,Blood, 99(10):3748-3755 (2002).

In certain embodiments, the components of the invention may be isolated.Generally, the term “isolated” means separated from constituents,cellular and otherwise, in which the polynucleotide, peptide,polypeptide, protein, antibody, or fragments thereof, are normallyassociated with in nature. As used herein, a peptide can be defined asany compound consisting of two or more amino acids. The skilled artisanwill understand that a peptide and a polypeptide and protein may be usedinterchangeably when describing the components of the invention. Apolypeptide is generally a chain of peptides or amino acids that isusually less than 100 amino acids long. A protein is a large moleculecomposed of one or more chains of amino acids in a specific order. As isapparent to those of skill in the art, a non-naturally occurringpolynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, does not require “isolation” to distinguish it from itsnaturally occurring counterpart. In addition, a “concentrated,”“separated” or “diluted” polynucleotide, peptide, polypeptide, protein,antibody, or fragment thereof is distinguishable from its naturallyoccurring counterpart in that the concentration or number of moleculesper volume is greater than (concentrated) or less than (separated) thanthat of its naturally occurring counterpart. A polynucleotide, peptide,polypeptide, protein, antibody, or fragment thereof, which differs fromthe naturally occurring counterpart in its primary sequence or forexample, by its glycosylation pattern, need not be present in itsisolated form. The compound is distinguishable from its naturallyoccurring counterpart by its primary sequence, or alternatively, byanother characteristic such as glycosylation pattern. Although notexplicitly stated for each of the inventions disclosed herein, it is tobe understood that all of the above embodiments for each of thecompositions disclosed below and under the appropriate conditions areprovided by this invention. Thus, as a non-limiting example, anon-naturally occurring polynucleotide is provided as a separateembodiment from the isolated naturally occurring polynucleotide.Furthermore, a protein produced in a bacterial cell is provided as aseparate embodiment from the naturally occurring protein isolated from aeukaryotic cell which produces it in nature.

Several peptide fragments of the CD19 and CD20 have the ability tostimulate a T-lymphocyte mediated cellular immune response. Peptides ofthe present invention can include the amino acid sequences of thesepeptide fragments in any configuration or location in the peptide. Insome embodiments, specific positions in the peptides, referred tohereinafter as anchor positions, have defined amino acids. Examples ofsuch anchor positions associated with strong HLA-A2.1 binding to thepeptide can include Leu or Ile at position 2, and/or Val at position 9.Residues putatively identified as strongly associated with weak HLA-A2.1binding at the anchor positions are Asp, Glu, or Pro at position 1; Aspor Glu at position 3; Arg, Lys, H is, or Ala at position 4; Pro atposition 5; Arg, Lys, or H is at position 7; Asp, Glu, Arg, Lys, or H isat position 8; and Arg, Lys, and/or H is at position 9. In someembodiments, the peptides disclosed herein do not have one, two, three,four or more of the above residues at the identified anchor positionsthat are associated with poor HLA-A2.1 binding. Generally, throughoutthis specification the one and three letter codes denoting amino acidresidues are used in accordance with standard naming conventions, suchas set forth by IUPAC.

In some embodiments, position 2 of the peptide L or I. In these andother embodiments, position 9, relative to the other anchor fragments,is V. Preferably, peptide fragments of the CD19 or CD20 protein havingone or more of these anchor positions at position 2 and/or position 9,such as those shown below, are used in the present methods andcompositions. Specifically, such peptide fragments can have thefollowing sequences, where a 2 indicates the presence of an anchor aminoacid at the second position as recited above, i.e., a L or I, and a 9indicates the presence of an anchor amino acid at the ninth position asrecited above, i.e., a V. An X indicates the presence of any amino acidresidue.

X2XXXXXX (SEQ ID NO: 15) XXXXXXXX9 (SEQ ID NO: 16) X2XXXXXX9 (SEQ ID NO:17)

In some embodiments, the present peptides will contain the motif LLF,LLFF (SEQ ID NO: 18), LLFL (SEQ ID NO: 19), FLLFL (SEQ ID NO: 20),FFLLFL (SEQ ID NO: 21), LLFFLL (SEQ ID NO: 22), LFFLLFL (SEQ ID NO: 23)or LLFFLLFL (SEQ ID NO: 24), particularly when the fragment is derivedfrom CD19. In other embodiments, the peptides can contain the motifsILS, MES, KMES (SEQ ID NO: 25), MESL (SEQ ID NO: 26), KMESL (SEQ ID NO:27) or LKMESL (SEQ ID NO: 28), particularly when the fragment is derivedfrom CD20.

The present peptides should be capable of stimulating a cytotoxicT-lymphocyte reaction. The anchor positions provide a guide to theskilled artisan for modifying fragments of CD19 or CD20 to increase theimmunogenicity of the peptide and “modify” the cytotoxic T-lymphocytereaction. As will be understood by one skilled in the art, generally,the first, second, and ninth amino acids in the peptide fragments areconsidered to be important for binding to MHC molecules and the third,fourth, fifth, sixth and seventh amino acids are considered as importantfor recognition by T cell receptors. However, the interactions withneighboring amino acids within the peptide are also important to MHCbinding and recognition by T cell receptors. As will be understood bythe skilled artisan, the anchor positions denoted above are onlynumbered with respect to one another the anchor positions can be placedin any relative orientation or appropriate place within a largerimmunogenic peptide. For example, any of these sequences can be part of25 amino acid peptide and anchor position 1 can start at position 5, 7,8, etc. within the larger peptide. Preferably, larger peptides havingthese anchor positions embedded within them have sequences at thenon-anchor positions that correspond to the CD19 or CD20 sequence. Thus,target fragments of CD19 and CD20 and mammalian homologs thereof can beidentified by aligning the desired anchor position or positions with theCD19 or CD20 or homologous protein sequence and selecting the desiredportion of the CD19 or CD20 sequence or homolog.

Specific examples of CD19 peptides include those containing thesequences: RLLFFLLFL (SEQ ID NO: 1), TLAYLIFCL (SEQ ID NO: 2), LLFLTPMEV(SEQ ID NO: 3), KLMSPKLYV (SEQ ID NO: 4), or LLFFLLFLV (SEQ ID NO: 5)with or without one or more conservative or nonconservative amino acidsubstitutions. Specific examples of CD20 peptides include thosecontaining the sequences: SLFLGILSV (SEQ ID NO: 6), AISGMILSI (SEQ IDNO: 7), FIRAHTPYI (SEQ ID NO: 8), SLNFIRAHT (SEQ ID NO: 9), LKMESLNFI(SEQ ID NO: 10), SHFLKMESL (SEQ ID NO: 11), or YLFLGILSV (SEQ ID NO: 12)with or without one or more conservative or nonconservative amino acidsubstitutions. Combinations of these peptides are also suitable for usein the present compounds and methods described herein. In someembodiments, the present peptides can also be made up of repeats of anyof the above sequences, combinations of the sequences or both.

Biologically functionally equivalent variants of the present CD19 orCD20 polypeptide fragments, i.e., variants of polypeptides which retainthe function of the natural polypeptide fragment, can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art. Examples of these methods may be such asmethods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook etal., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.)1989); or Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., New York. The skilledartisan will also realize that conservative amino acid substitutions canbe made in the present polypeptides to provide such functionally activehomologs of the forgoing polypeptides, i.e., the homologs retain thefunctional capabilities of the polypeptides. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Conservative substitutions of amino acids generally are understoodto include substitutions made amongst amino acids within the followinggroups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;(f) Q, N; and (g) E, D. Nevertheless, this grouping is meant to benon-limiting. The present invention also encompasses polypeptides withone or more nonconservative amino acid substitutions that retain similarfunctionality as compared to the non-modified peptide. Generally a“nonconservative amino acid substitution” is understood to be an aminoacid substituted by an alternative amino acid of differing chargedensity, hydrophilicity/hydrophobicity, size, and/or configuration(e.g., Val for Phe). The means of making such modifications are wellknown in the art and can be readily accomplished by means ofcommercially available kits and vectors (e.g., New England Biolabs,Inc., Beverly, Mass.; Clontech, Palo Alto, Calif.). Moreover, the meansof assessing such substitutions (e.g., in terms of effect on ability tobind and enter cells) are known in the art and described for example inU.S. Pat. No. 6,329,190.

The polypeptides of the present invention also include variants of theCD19 or CD20 fragments that retain the ability to stimulate a Th1 or CTLimmune response in PBMCs. Such variants may include various structuralforms of the primary protein, including related and homologous proteinsthat can be found in non-human species. Due to the presence of ionizableamino and carboxyl groups, for example, a polypeptide fragment can be inthe form of an acidic or basic salt, or it can be in neutral form.Individual amino acid residues can also be modified by oxidation orreduction.

Variants within the scope of this invention also include polypeptides inwhich the primary amino acid structure of the polypeptide fragment ismodified by forming covalent or aggregative conjugates with otherpolypeptides or chemical moieties such as glycosyl groups, lipids,phosphate, acetyl groups and the like. Covalent derivatives can beprepared, for example, by linking particular functional groups to aminoacid side chains or at the N- or C-termini. Alternatively, forderivatives in which an additional polypeptide is joined to a CD19 orCD20 fragment, a fusion protein can be prepared using recombinant DNAtechniques, such as described below. As will be understood by theskilled artisan, fusion peptides containing the 8-12 amino acid CD19 orCD20 fragment of the present invention are not limited to 8-12 aminoacids in total size, but instead set forth only that the immunogenicfragment of the CD19 or CD20 antigen is 8-12 residues in size. In onesuch embodiment, the CD19 or CD20 polypeptide can be conjugated to asignal (or leader) polypeptide sequence at the N-terminal region of theCD19 or CD20 protein which co-translationally or post-translationallydirects transfer of the protein from its site of synthesis to a site offunction inside or outside of the cell membrane or wall (e.g., the yeastα-factor leader).

Also provided by this application are the polypeptides and proteinsdescribed herein conjugated to a detectable agent for use in diagnosticmethods. For example, detectably labeled proteins and polypeptides canbe bound to a column and used for the detection and purification ofantibodies. They also are useful as immunogens for the production ofantibodies as described below. Furthermore, the proteins and fragmentsof this invention are useful in an in vitro assay system to screen foragents or drugs that modulate cellular processes. This assay system isdescribed in more detail in a later paragraph.

Detectable agents used in the diagnostic methods include fusionsproteins that facilitate purification or identification of thepolypeptides (e.g., poly-His). However, as the skilled artisanunderstands these types of detectable agent are meant to illustrate anon-limiting example. For instance, the peptide described by Hopp etal., Bio/Technol 6:1204 (1988), is a highly antigenic peptide that canbe used to facilitate identification. Such a peptide provides an epitopereversibly bound by a specific monoclonal antibody, enabling rapid assayand facile purification of expressed recombinant protein. The peptide ofHopp et al. contains a sequence that may be specifically cleaved bybovine mucosal enterokinase, allowing removal of the peptide from thepurified CD19 or CD20 fragment. As another advantage, fusion proteinscapped with such peptides can be resistant to intracellular degradationin E. coli.

Protein fusions encompassed by this invention further include, forexample, the polypeptides linked to an immunoglobulin Fc region. Forexample, if fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form a protein oligomer with as many asfour CD19 or CD20 protein fragment regions. Also within the scope of thepresent invention are fusion proteins consisting of CD19 and CD20fragments fused to polypeptides linked to a leucine zipper domain.Leucine zipper domains are described, for example, in published PCTApplication WO 94/10308. The present polypeptides comprising leucinezippers may, for example, be oligomeric, dimeric or trimeric. All of theabove protein fusions can be prepared by chemical linkage or as fusionproteins, as described in U.S. Pat. No. 6,013,268. Preferred proteinfusions include polypeptides that comprise sequences useful forstimulating immunity to infectious pathogens (e.g., antigens). Suchsequences can be derived, for example, from viruses, tumor cells,parasites or bacteria.

The proteins and polypeptides of this invention can be obtained bychemical synthesis using a commercially available automated peptidesynthesizer such as those manufactured by Perkin Elmer/AppliedBiosystems, Inc., Model 430A or 431A, Foster City, Calif. Thesynthesized protein or polypeptide can be precipitated and furtherpurified, for example by high performance liquid chromatography (HPLC).Accordingly, this invention also provides a process for chemicallysynthesizing the proteins of this invention by providing the sequence ofthe protein and reagents, such as amino acids and enzymes and linkingtogether the amino acids in the proper orientation and linear sequence.

Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods using the host cell and vector systemsdescribed throughout this specification.

Nonpeptide analogs of peptides, e.g., structures provide an increase instabilization or lessened biodegradation, are also contemplated. Peptidemimetic analogs can be prepared based on a selected binding peptide byreplacement of one or more amino acid residues by nonpeptide moieties.Preferably, the nonpeptide moieties permit the peptide to retain itsnatural conformation, and/or stabilize a preferred, e.g., bioactive,confirmation. Peptides containing nonpeptide moieties can be tested inmolecular or cell-based binding assays to assess the effect of thesubstitution(s) on conformation and/or activity. One example of methodsfor preparation of nonpeptide mimetic analogs from peptides is describedin Nachman et al., Regul Pept 57:359-370 (1995), and disclosed in U.S.Pat. No. 6,291,430.

The proteins of this invention also can be combined with various liquidphase carriers, such as sterile or aqueous solutions, pharmaceuticallyacceptable carriers, suspensions and emulsions. Examples of non-aqueoussolvents that may be combined with the proteins of this inventioninclude propyl ethylene glycol, polyethylene glycol and vegetable oils.When used to prepare antibodies, the carriers also can include anadjuvant that is useful to non-specifically augment a specific immuneresponse. In a particular situation, a skilled artisan can easilydetermine whether an adjuvant is required and select one. However, forthe purpose of illustration only, suitable adjuvants include, but arenot limited to Freund's Complete and Incomplete, mineral salts,polynucleotides, GM-CSF and Keyhole Limpet Hemocyanin (KLH). Adjuvantcan be used as is known in the art.

II. Nucleic Acids/Polynucleotides/Nucleotides/Fragments Thereof

Polynucleotides of the subject invention generally comprise a DNA or RNAsequence that encodes all or a portion of the above CD19 or CD20polypeptide fragments, or that is complementary to such a sequence.Nucleic acids may encode proteins related or homologous to the CD19 orCD20 polypeptides discussed above. Preferably, the CD19 or CD20nucleotide fragment is a lymphoma associated nucleic acid or is anucleic acid or polypeptide expressed preferentially in lymphomas.Various methods for determining the expression of a nucleic acid and/ora polypeptide in normal and leukemia cells are known to those of skillin the art.

The reported nucleic acid sequence encoding the CD19 protein describedabove, as reported in Tedder et al., J Immunol, 143(2):712-717 (1989),is (SEQ ID NO: 29):

   1 GAATTCCTCT GACCACCATG CCACCTCCTC GCCTCCTCTT CTTCCTCCTC TTCCTCACCC  62 CCATGGAAGT CAGGCCCGAG GAACCTCTAG TGGTGAAGGT GGAAGAGGGA GATAACGCTG 121 TGCTGCAGTG CCTCAAGGGG ACCTCAGATG GCCCCACTCA GCAGCTGACC TGGTCTCGGG 181 AGTCCCCGCT TAAACCCTTC TTAAAACTCA GCCTGGGGCT GCCAGGCCTG GGAATCCACA 241 TGAGGCCCCT GGCATCCTGG CTTTTCATCT TCAACGTCTC TCAACAGATG GGGGGCTTCT 301 ACCTGTGCCA GCCGGGGCCC CCCTCTGAGA AGGCCTGGCA GCCTGGCTGG ACAGTCAATG 361 TGGAGGGCAG CGGGGAGCTG TTCCGGTGGA ATGTTTCGGA CCTAGGTGGC CTGGGCTGTG 421 GCCTGAAGAA CAGGTCCTCA GAGGGCCCCA GCTCCCCTTC CGGGAAGCTC ATGAGCCCCA 481 AGCTGTATGT GTGGGCCAAA GACCGCCCTG AGATCTGGGA GGGAGAGCCT CCGTGTGTCC 541 CACCGAGGGA CAGCCTGAAC CAGAGCCTCA GCCAGGACCT CACCATGGCC CCTGGCTCCA 601 CACTCTGGCT GTCCTGTGGG GTACCCCCTG ACTCTGTGTC CAGGGGCCCC CTCTCCTGGA 661 CCCATGTGCA CCCCAAGGGG CCTAAGTCAT TGCTGAGCCT AGAGCTGAAG GACGATCGCC 721 CGGCCAGAGA TATGTGGGTA ATGGAGACGG GTCTGTTGTT GCCCCGGGCC ACAGCTCAAG 781 ACGCTGGAAA GTATTATTGT CACCGTGGCA ACCTGACCAT GTCATTCCAC CTGGAGATCA 841 CTGCTCGGCC AGTACTATGG CACTGGCTGC TGAGGACTGG TGGCTGGAAG GTCTCAGCTG 901 TGACTTTGGC TTATCTGATC TTCTGCCTGT GTTCCCTTGT GGGCATTCTT CATCTTCAAA 961 GAGCCCTGGT CCTGAGGAGG AAAAGAAAGC GAATGACTGA CCCCACCAGG AGATTCTTCA1021 AAGTGACGCC TCCCCCAGGA AGCGGGCCCC AGAACCAGTA CGGGAACGTG CTGTCTCTCC1081 CCACACCCAC CTCAGGCCTC GGACGCGCCC AGCGTTGGGC CGCAGGCCTG GGGGGCACTG1141 CCCCGTCTTA TGGAAACCCG AGCAGCGACG TCCAGGCGGA TGGAGCCTTG GGGTCCCGGA1201 GCCCGCCGGG AGTGGGCCCA GAAGAAGAGG AAGGGGAGGG CTATGAGGAA CCTGACAGTG1261 AGGAGGACTC CGAGTTCTAT GAGAACGACT CCAACCTTGG GCAGGACCAG CTCTCCCAGG1321 ATGGCAGCGG CTACGAGAAC CCTGAGGATG AGCCCCTGGG TCCTGAGGAT GAAGACTCCT1381 TCTCCAACGC TGAGTCTTAT GAGAACGAGG ATGAAGAGCT GACCCAGCCG GTCGCCAGGA1441 CAATGGACTT CCTGAGCCCT CATGGGTCAG CCTGGGACCC CAGCCGGGAA GCAACCTCCC1501 TGGGGTCCCA GTCCTATGAG GATATGAGAG GAATCCTGTA TGCAGCCCCC CAGCTCCACT1561 CCATTCGGGG CCAGCCTGGA CCCAATCATG AGGAAGATGC AGACTCTTAT GAGAACATGG1621 ATAATCCCGA TGGGCCAGAC CCAGCCTGGG GAGGAGGGGG CCGCATGGGC ACCTGGAGCA1681 CCAGGTGATC CTCAGGTGGC CAGCCTGGAT CTCCTCAAGT CCCCAAGATT CACACCTGAC1741 TCTGAAATCT GAAGACCTCG AGCAGATGAT GCCAACCTCT GGAGCAATGT TGCTTAGGAT1801 GTGTGCATGT GTGTAAGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT ATACATGCCA1861 GTGACACTTC CAGTCCCCTT TGTATTCCTT AAATAAACTC AATGAGCTCT TCCAATCCAA1921 AAATGTTAAA ATTAGCCAGG CATAGTTGTG TGTGCCTACA GTGCTACAGG AGGCTGAGGC1981 AAGAGGATTG CTTGAGTTAA GGAAGGAAGT CAAGGCTGCA GTGAGCTATG GTCATGCCAC2041 TGCACTCCAG CCTGGGCAAC AGCAAGACCC TGTGTCCAAA AAAAAAAAAG GAATTC

The reported nucleic acid sequence encoding the CD20 protein describedabove is (SEQ ID NO: 30):

   1 AAAGACAAAC TGCACCCACT GAACTCCGCA GCTAGCATCC AAATCAGCCC TTGAGATTTG  61 AGGCCTTGGA GACTCAGGAG TTTTGAGAGC AAAATGACAA CACCCAGAAA TTCAGTAAAT 121 GGGACTTTCC CGGCAGAGCC AATGAAAGGC CCTATTGCTA TGCAATCTGG TCCAAAACCA 181 CTCTTCAGGA GGATGTCTTC ACTGGTGGGC CCCACGCAAA GCTTCTTCAT GAGGGAATCT 241 AAGACTTTGG GGGCTGTCCA GATTATGAAT GGGCTCTTCC ACATTGCCCT GGGGGGTCTT 301 CTGATGATCC CAGCAGGGAT CTATGCACCC ATCTGTGTGA CTGTGTGGTA CCCTCTCTGG 361 GGAGGCATTA TGTATATTAT TTCCGGATCA CTCCTGGCAG CAACGGAGAA AAACTCCAGG 421 AAGTGTTTGG TCAAAGGAAA AATGATAATG AATTCATTGA GCCTCTTTGC TGCCATTTCT 481 GGAATGATTC TTTCAATCAT GGACATACTT AATATTAAAA TTTCCCATTT TTTAAAAATG 541 GAGAGTCTGA ATTTTATTAG AGCTCACACA CCATATATTA ACATATACAA CTGTGAACCA 601 GCTAATCCCT CTGAGAAAAA CTCCCCATCT ACCCAATACT GTTACAGCAT ACAATCTCTG 661 TTCTTGGGCA TTTTGTCAGT GATGCTGATC TTTGCCTTCT TCCAGGAACT TGTAATAGCT 721 GGCATCGTTG AGAATGAATG GAAAAGAACG TGCTCCAGAC CCAAATCTAA CATAGTTCTC 781 CTGTCAGCAG AAGAAAAAAA AGAACAGACT ATTGAAATAA AAGAAGAAGT GGTTGGGCTA 841 ACTGAAACAT CTTCCCAACC AAAGAATGAA GAAGACATTG AAATTATTCC AATCCAAGAA 901 GAGGAAGAAG AAGAAACAGA GACGAACTTT CCAGAACCTC CCCAAGATCA GGAATCCTCA 961 CCAATAGAAA ATGACAGCTC TCCTTAAGTG ATTTCTTCTG TTTTCTGTTT CCTTTTTTAA1021 ACATTAGTGT TCATAGCTTC CAAGAGACAT GCTGACTTTC ATTTCTTGAG GTACTCTGCA1081 CATACGCACC ACATCTCTAT CTGGCCTTTG CATGGAGTGA CCATAGCTCC TTCTCTCTTA1141 CATTGAATGT AGAGAATGTA GCCATTGTAG CAGCTTGTGT TGTCACGCTT CTTCTTTTGA1201 GCAACTTTCT TACACTGAAG AAAGGCAGAA TGAGTGCTTC AGAATGTGAT TTCCTACTAA1261 CCTGTTCCTT GGATAGGCTT TTTAGTATAG TATTTTTTTT TGTCATTTTC TCCATCAGCA1321 ACCAGGGAGA CTGCACCTGA TGGAAAAGAT ATATGACTGC TTCATGACAT TCCTAAACTA1381 TCTTTTTTTT ATTCCACATC TACGTTTTTG GTGGAGTCCC TTTTTATCAT CCTTAAAACA1441 ATGATGCAAA AGGGCTTTAG AGCACAATGG ATCT

The nucleic acids encompassed by the invention contemplate thedegeneracy of the genetic code in which nucleic acids can be coded byalternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets canbe employed to direct the protein synthesis apparatus, in vivo or invitro, to incorporate a serine residue. Biological molecules may also bedifferent when isolated. Similarly, nucleotide sequence triplets whichencode other amino acid residues include, but are not limited to: CCA,CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG(arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT(asparagine codons); and ATA, ATC and ATT (isoleucine codons). Asunderstood by the skilled artisan, other amino acid residues can beencoded similarly by multiple nucleotide sequences. Thus, the inventionembraces degenerate nucleic acids that differ from one or more or thebiologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code. Further contemplated by the inventionare antisense oligonucleotides that selectively bind to a leukemiaassociated gene nucleic acid molecule. Additionally, nucleic acidmimetics, such as peptide nucleic acids are contemplated in thedefinition of nucleic acids.

The disclosed polynucleotides and peptides can be used for comparison toknown and unknowns sequences using a computer-based method to match asample sequence with known sequences. Thus, this invention also providesthe polynucleotides or peptides in a computer database or in computerreadable form, including applications utilizing the internet.

A linear search through such a database can be used. Alternatively, thepolynucleotide sequence can be converted into a unique numericrepresentation. The comparison aspects can be implemented in hardware orsoftware, or a combination of both. Preferably, these aspects of theinvention are implemented in computer programs executing on aprogrammable computer comprising a processor, a data storage system(including volatile and non-volatile memory and/or storage elements), atleast one input device, and at least one output device. Data inputthrough one or more input devices for temporary or permanent storage inthe data storage system includes both polypeptide and polynucleotidesequences, and can include previously generated polynucleotides,polypeptides and related codes for known and/or unknown sequences.Program code is applied to the input data to perform the functionsdescribed above and generate output information. The output informationis applied to one or more output devices, in known fashion.

Each such computer program is preferably stored on a storage media ordevice (e.g., ROM or magnetic diskette) readable by a general or specialpurpose programmable computer for configuring and operating the computerwhen the storage media or device is read by the computer to perform theprocedures described herein. The inventive system can also be consideredto be implemented as a computer-readable storage medium, configured witha computer program, where the storage medium so configured causes acomputer to operate in a specific and predefined manner to perform thefunctions described herein.

The polynucleotides of the present invention also can serve as primersfor the detection of genes or gene transcripts that are expressed inantigen presenting cells, for example, to confirm entry of thepolynucleotides into host cells by amplification or other methods knownin the art. In this context, amplification means any method employing aprimer-dependent polymerase capable of replicating a target sequencewith reasonable fidelity. Amplification can be carried out by natural orrecombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragmentof E. coli DNA polymerase, and reverse transcriptase.

The invention further provides the polynucleotide of interestoperatively linked to a promoter of nucleotide, including RNAtranscription, as well as other regulatory sequences for replicationand/or transient or stable expression. As used herein, the term“operatively linked” means positioned in such a manner that the promoterwill direct transcription off the nucleotide of interest. Examples ofpromoters that may be used “include” SP6, T4 and T7. In certainembodiments, cell-specific promoters are used for cell-specificexpression of the polynucleotide of interest. Vectors which contain apromoter or a promoter/enhancer, with termination codons and selectablemarker sequences, as well as a cloning site into which an inserted pieceof DNA can be operatively linked to that promoter are well known in theart and commercially available. For general methodology and cloningstrategies, (see Gene Expression Technology, Goeddel ed., AcademicPress, Inc. (1991), and references cited therein; and Vectors: EssentialData Series, Gacesa and Ramji, eds., John Wiley & Sons, NY (1994)).These references contain maps, functional properties, commercialsuppliers and a reference to GenEMBL accession numbers for varioussuitable vectors. Preferable, these vectors are capable of transcribingRNA in vivo or in vitro.

Expression vectors containing the polynucleotides are useful to obtainhost vector systems that produce proteins and polypeptides. It isimplied that these expression vectors must be replicable in the hostorganisms either as episomes or as an integral part of the chromosomalDNA. Suitable expression vectors include plasmids, viral vectors,including adenoviruses, adeno-associated viruses, retroviruses, cosmids,etc. Adenoviral vectors are particularly useful for introducing genesinto tissues in vivo because of their high levels of expression andefficient transformation of cells both in vivo and in vitro. When usingthese vectors, and/or a nucleic acid is inserted into a suitable hostcell, e.g., a prokaryotic or a eukaryotic cell and the host cellreplicates, the protein can be recombinantly produced. Suitable hostcells will depend on the vector and can include mammalian cells, animalcells, human cells, simian cells, insect cells, yeast cells, andbacterial cells. See Sambrook et al., 1989, supra. In addition to theuse of the above vectors, including viral vectors for insertion ofexogenous nucleic acid into cells, the nucleic acid can be inserted intothe host cell by other methods well known in the art such astransformation for bacterial cells; transfection, for example by calciumphosphate precipitation, for mammalian cells; or DEAE-dextran;electroporation; or microinjection for mammalian cells. Thesemethodologies are well known in the art and are demonstrated inSambrook. See Sambrook et al., 1989, supra, for this methodology. Thus,this invention also provides a host cell, e.g., a mammalian cell, ananimal cell (rat or mouse), a human cell, or a prokaryotic cell such asa bacterial cell, containing a polynucleotide encoding a protein orpolypeptide or antibody.

When vectors are used for gene therapy in vivo or ex vivo, apharmaceutically acceptable vector is preferred, such as areplication-incompetent retroviral or adenoviral vector.Pharmaceutically acceptable vectors containing the nucleic acids of thisinvention can be further modified for transient or stable expression ofthe inserted polynucleotide. As used herein, the term “pharmaceuticallyacceptable vector” includes, but is not limited to, a vector or deliveryvehicle having the ability to selectively target and introduce thenucleic acid into dividing cells. An example of such a vector is a“replication-incompetent” vector defined by its inability to produceviral proteins, precluding spread of the vector's biological material inthe infected host cell. An example of a replication-incompetentretroviral vector is LNL6 (Miller A D et al., BioTechniques 7:980-990(1989)). The methodology of using replication-incompetent retrovirusesfor retroviral-mediated gene transfer of gene markers is wellestablished (Correll et al., Proc Natl Acad Sci USA 86:8912 (1989);Bordignon, Proc Natl Acad Sci USA, 86:8912-8952 (1989); Culver K, ProcNatl Acad Sci USA 88:3155 (1991); and Rill D R, Blood, 79(10):2694-2700(1991)) and will be understood by one of skill in the art.

The host cells containing the polynucleotides of this invention areuseful for the recombinant replication of the polynucleotides and forthe recombinant production of peptides. Alternatively, the cells can beused to induce an immune response in a subject in the methods describedherein. When the host cells are antigen presenting cells, they can beused to expand a population of immune effector cells such as tumorinfiltrating lymphocytes, which in turn are useful in adoptiveimmunotherapies.

III. Protein Binding Agents (Antibodies)

The invention also involves agents which bind to the leukemia associatedpolypeptides disclosed herein. Such binding agents can be used inscreening assays to detect the presence or absence of the polypeptides.The binding agents may also be used in purification protocols to isolatethese polypeptides. Such binding partners can be used further toselectively target drugs, toxins or other molecules to leukemia cellswhich present the associated polypeptides. In this manner, cells presentin cells which express the CD19 or CD20 fragments can be treated withcytotoxic compounds.

The invention, therefore, involves antibodies or fragments of antibodieshaving the ability to selectively bind to the disclosed polypeptides.Antibodies against the CD19 or CD20 antigens are discussed above.Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology. The antibodies can include, butare not limited to mouse, rat, rabbit, and/or human antibodies. Theantibodies are useful to identify and purify polypeptides and APCsexpressing the polypeptides.

The antibodies of the present invention are prepared by any of a varietyof methods, including administering protein, fragments of protein, cellsexpressing the protein or fragments thereof and the like to an animal toinduce polyclonal antibodies. Laboratory methods for producingpolyclonal antibodies and monoclonal antibodies, as well as deducingtheir corresponding nucleic acid sequences, are known in the art, forexample, see Harlow and Lane, 1988, supra, and Sambrook et al, 1989,supra. The monoclonal antibodies of this invention can be biologicallyproduced by introducing protein or a fragment thereof into an animal,e.g., a mouse or a rabbit. The antibody producing cells in the animalare then isolated and fused with myeloma cells or heteromyeloma cells toproduce hybrid cells or hybridomas. Accordingly, hybridoma cellsproducing the monoclonal antibodies of this invention also are provided.

The antibodies of this invention can be linked to a detectable agent orlabel. There are many different labels and methods of labeling known tothose of ordinary skill in the art. The coupling of antibodies to lowmolecular weight haptens can increase the sensitivity of variousbiological assays. The haptens can then be specifically detected bymeans of a second reaction. For example, it is common to use haptenssuch as biotin, which reacts with avidin, or dinitropherryl, pyridoxal,and fluorescein, which can react with specific anti-hapten antibodies.See Harlow and Lane, 1988, supra. Antibodies also can be coupled tospecific labeling agents for imaging or to antitumor agents, including,but not limited to, methotrexate, radioiodinated compounds, toxins suchas ricin, other cytostatic or cytolytic drugs, and so forth. Antibodiesprepared according to the invention preferably are specific for the CD19or CD20 complexes described herein.

The monoclonal antibodies of the invention also can be bound to manydifferent carriers. Thus, this invention provides compositionscontaining the antibodies and another substance, which may be active orinert. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

Thus, using the protein or fragment thereof, and well known methods, oneof skill in the art can produce and screen the hybridoma cells andantibodies of this invention for antibodies having the ability to bindthe proteins or polypeptides. As detailed herein, such antibodies canalso be used to identify tissues expressing protein or to purifyprotein.

If a monoclonal antibody being tested binds with the protein orpolypeptide, then the antibody being tested and the antibodies providedby the hybridomas of this invention are equivalent. It also is possibleto determine without undue experimentation, whether an antibody hasoverlapping specificity with the monoclonal antibody of this inventionby determining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

The term “antibody” is intended to include antibodies of all isotypes.Particular isotypes of a monoclonal antibody can be prepared eitherdirectly by selecting from the initial fusion, or prepared secondarily,such as from a parental hybridoma secreting a monoclonal antibody ofdifferent isotype by using the sib selection technique to isolate classswitch variants. This procedure is described in Steplewski et al., ProcNatl Acad Sci, 82:8653 (1985); and Spira et al., J Immunol Methods,74:307 (1984).

This invention also contemplates the use of biological active fragmentsof the polyclonal and monoclonal antibodies described above. These“antibody fragments” retain some ability to selectively bind with theirantigen or immunogen. Such antibody fragments can include, but are notlimited to: (1) Fab, (2) Fab′, (3) F(ab′)₂, (4) Fv, and (5) SCA. Aspecific example of “a biologically active antibody fragment” is a CDRregion of an antibody. Methods of making these antibody fragments areknown in the art, see for example, Harlow and Lane, 1988, supra.

The isolation of hybridomas secreting monoclonal antibodies with thespecificity of the monoclonal antibodies of the invention can beaccomplished by the production of one of ordinary skill in the artanti-idiotypic antibodies (Herlyn et al., Science, 232:100 (1986)). Ananti-idiotypic antibody is an antibody that recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest. Generally, idiotypic identity between monoclonalantibodies of two hybridomas demonstrates that the two monoclonalantibodies are the same with respect to their recognition of the sameepitopic determinant. Thus, by using antibodies to the epitopicdeterminants on a monoclonal antibody it is possible to identify otherhybridomas expressing monoclonal antibodies of the same epitopicspecificity.

Compositions containing the antibodies, fragments thereof or cell lineswhich produce the antibodies, are encompassed by this invention. Whenthese compositions are to be used pharmaceutically, they can be combinedwith a pharmaceutically acceptable carrier.

IV. Pulsing Antigen Presenting Cells

The polypeptides of this invention can be pulsed into antigen presentingcells either in vivo or in vitro using the methods described herein.Various methods of pulsing antigen presenting cells are disclosed inU.S. Pat. No. 6,306,640; Lodge et al., Cancer Res, 60:829 (2000); Lau etal., J Immun, 24 (1):66 (2001); Gajewski et al., Clin Cancer Res, 7:895(2001); Morse et al., Clin Cancer Res, 5:1331 (1999); and Schmidt etal., Proc Natl Acad Sci, 94:3262 (1997). Antigen-presenting cells,include, but are not limited to dendritic cells (DCs),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-immunostimulatory molecules. The methods describedbelow focus primarily on DCs which are the most potent, preferred APCs.These antigen presenting host cells containing the polypeptides orproteins are further provided.

The term “antigen-presenting cells” or “APCs” includes both intact,whole cells as well as other molecules that are capable of inducing thepresentation of one or more antigens, preferably in association with MHCmolecules. Examples of suitable APCs are discussed in detail below andinclude, but are not limited to, whole cells such as macrophages,dendritic cells, B cells, purified MHC class I molecules complexed tobeta 2-microglobulin and foster antigen presenting cells.

Dendritic cells(DCs) are the most effective type of antigen presentingcells (APC) in the human body. Dendritic cells express significantlevels of co-immunostimulatory (CD86, CD80) and MHC class I and class IImolecules on their cell surface. Many different factors and cytokinecombinations have been demonstrated to produce mature dendritic cells(mDCs) in vitro. It has been shown that DCs provide all the signals,which are generally characterized into two types, required for T cellactivation and proliferation. The first type of signal, which givesspecificity to the immune response, is mediated through interactionbetween the T cell receptor/CD3 (“TCR/CD3”) complex and an antigenicpeptide presented by a major histocompatibility complex (“MHC”) class Ior II protein on the surface of DCs and other APCs. This interactionbetween TCR/CD3 and the antigenic peptide is necessary, but notsufficient, for T cell activation to occur. In fact, without the secondtype of signal, the signal between the TCR/CD3 and Antigenic peptideresult in T cell energy. The second type of signal, called aco-immunostimulatory signal, is neither antigen-specific norMHC-restricted, this second type of signal, and can lead to a fullproliferation response of T cells and induction of T cell effectorfunctions in the presence of the first type of signals. As used herein,“dendritic cell” is to include, but not be limited to a pulsed dendriticcell, a foster cell or a dendritic cell hybrid.

As demonstrated in the Examples, the present methods, peptides andeffector cells can induce or provide not only significant levels ofcytotoxicity, but levels of cytotoxicity from about 10 percent up toabout 50 percent, or more specifically 21, 29, 31, 33, 34, 35, 36, 37 or39 percent to 42, 43, 45, 46, 50 percent in various cell types.

Isolated antigen presenting host cells that present the polypeptides ofthis invention in the context of MHC molecules are further useful toexpand and isolate a population of educated, antigen-specific immuneeffector cells. The term “immune effector cells” refers to cells capableof binding an antigen or mediating an immune response. These cellsinclude, but are not limited to, T cells, B cells, monocytes,macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for exampleCTL lines, CTL clones, and CTLs from tumor inflammatory regions, orother infiltrates. The immune effector cells, e.g., cytotoxic Tlymphocytes, are preferably produced by culturing naive immune effectorcells with antigen-presenting cells that present the polypeptides in thecontext of MHC molecules on the surface of the APCs. A “naive” cell is acell that has never been exposed to an antigen. The population ofeducated, antigen—specific immune effector cells can be purified usingmethods known in the art, e.g., FACS analysis or ficoll gradients. Themethods used to generate and culture the immune effector cells as wellas the populations produced thereby also are the inventor's contributionand invention. [Pharmaceutical compositions comprising the cells andpharmaceutically acceptable carriers are useful in adoptiveimmunotherapy. Prior to administration in vivo, the immune effectorcells can be screened in vitro for their ability to lyse melanoma tumorcells.]

In one embodiment, the immune effector cells and/or the APCs aregenetically modified. Using standard gene transfer, genes coding forco-immunostimulatory molecules and/or stimulatory cytokines can beinserted prior to, concurrent to or subsequent to expansion of theimmune effector cells.

The present invention also encompasses immune effector cells that havebeen exposed to polypeptides of the present invention, preferably wherethe polypeptides are in an isolated form. Alternative to the above, theimmune effector cells can be exposed to the polypeptides, preferably inthe presence of one or more stimulatory molecules, without the help ofantigen presenting cells.

V. Immune Response Induction

This invention also provides methods of inducing an immune response in asubject, comprising administering to the subject an effective amount ofthe polypeptides described above under the conditions that induce animmune response to the polypeptide. The polypeptides can be administeredin formulations or as polynucleotides encoding the polypeptides. Thepolynucleotides can be administered in a gene delivery vehicle or byinserting into a host cell that in turn recombinantly transcribes,translates and processes the encoded polypeptide. Isolated host cellscontaining the polynucleotides of this invention and a pharmaceuticallyacceptable carrier can therefore be combined with an appropriate andeffective amount of an adjuvant, cytokine or co-immunostimulatorymolecule for an effective vaccine regimen. The vaccination can either beprophylactic or for treatment of established cancer. In one embodiment,the host cell used in the vaccine regimen is an APC such as a dendriticcell. In some embodiments, host cell can be further modified byinserting a polynucleotide coding for an effective amount of either orboth of a cytokine or co-immunostimulatory molecule.

Co-administering an effective amount of a cytokine orco-immunostimulatory molecule with the methods of the invention. As usedherein, the term “cytokine” refers to any one of the numerous factorsthat exert a variety of effects on cells, for example, inducing growthor proliferation. Non-limiting examples of cytokines which can be usedalone or in combination in the practice of the present inventioninclude, interleukin-2 (IL-2), stem cell factor (SCF), interleukin 3(IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), granulocyte colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interleukin-1 alpha (IL-1.sub.I), interleukin-11(IL-11), MIP-1_(I), leukemia inhibitory factor (LIF), c-kit ligand,thrombopoietin (TPO) and flt3 ligand. The present invention alsoanticipates use of culture conditions in which one or more of the abovecytokines is specifically excluded from the medium. Generally, thesecytokines are commercially available from several vendors such as, forexample, Genzyme (Framingham, Mass.), Genentech (South San Francisco,Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems and Immunex(Seattle, Wash.). It is intended, although not always explicitly stated,that molecules having similar biological activity as wild-type orpurified cytokines (e.g., recombinantly produced or muteins thereof) maybe used while still maintaining within the spirit and scope of theinvention.

Co-immunostimulatory molecules may also be used in the presentinvention. “Co-immunostimulatory molecules” are involved in theinteraction between receptor-ligand pairs expressed on the surface ofantigen presenting cells and T cells. One exemplary receptor-ligand pairis the B7 co-immunostimulatory molecule on the surface of DCs and itscounter-receptor CD28 or CTLA-4 on T cells (Freeman et al., Science,262:909-991 (1993); Young et al., J Clin Invest, 90:229 (1992); Nabaviet al., Nature, 360:266 (1992)). Other important co-immunostimulatorymolecules are CD40, CD54, CD80, CD86.

Patient T cell assays can generally be performed by treating patientPBMCs with the reactive antigens and analyzing the cells for a suitableresponse. For example, the PBMC supernatant can be assayed for the levelof secreted cytokines. Preferably, the cytokine assayed isinterferon-gamma, (ICN-y) IL-2, IL-12 (either the p40 subunit orbiologically active p70), IL-1 or tumor necrosis factor-α TNF-α. Thecytokines interleukin-4 and interleukin-10 can also be assayed, sincethe levels of these representative Th2-type cytokines generally decreasein response to treatment with a polypeptide as described herein.Cytokines can be assayed, for example, in an ELISA format usingcommercially available antibodies specific for the cytokine of interest.Generally, positive results will be determined according to themanufacturer's instructions. Suitable antibodies can be obtained frommany commercial suppliers including Chemicon, Temucula, Calif. andPharMingen, San Diego, Calif. Alternatively, the treated PBMCs can beassayed for mRNA encoding one or more of the cytokines interferon-gamma,interleukin-2, interleukin-12 p40 subunit, interleukin-1 or tumornecrosis factor-α, or the PBMCs can be assayed for a proliferativeresponse as described herein. Alternatively, cytokines can be measuredby testing PBMC supernatants for cytokine-specific biologicalactivities.

VI. Method of Diagnosis

According to one aspect of the invention, methods for diagnosing adisorder that is characterized by expression of a CD19 or CD20 nucleicacid or polypeptide are provided. The methods involve contacting abiological sample isolated from a subject with an agent specific for theleukemia associated nucleic acid or polypeptide to detect the presenceof the leukemia associated nucleic acid or polypeptide in the biologicalsample. As used herein, “contacting” means placing the biological samplein sufficient proximity to the agent and under the appropriateconditions, e.g., concentration, temperature, time, ionic strength, etc.to allow the specific interaction between the agent and the leukemiaassociated nucleic acid or polypeptide that are present in thebiological sample. In general, the conditions for contacting the agentwith the biological sample are conditions known by those of ordinaryskill in the art to facilitate a specific interaction between a moleculeand its cognate (e.g., a protein and its receptor cognate, an antibodyand its protein antigen cognate, a nucleic acid and its complementarysequence cognate) in a biological sample. Exemplary conditions forfacilitating a specific interaction between a molecule and its cognateare described in U.S. Pat. No. 5,108,921, issued to Low et al.

The biological sample can be located in vivo or in vitro. For example,the biological sample can be a hematopoietic tissue in vivo and theagent specific for the leukemia associated nucleic acid or polypeptidecan be used to detect the presence of such molecules in thehematopoietic tissue (e.g., for imaging portions of the hematopoietictissue that express the leukemia associated genes and gene products).Alternatively, the biological sample can be located in vitro (e.g., ablood sample, a bone marrow biopsy, a tissue extract). In a particularlypreferred embodiment, the biological sample can be a cell-containingsample, more preferably a sample containing hematopoietic cells.

Although the method is not meant to be limiting, the skilled artisan candetermine which HLA molecule binds to the CD19 or CD20 fragments byexperiments utilizing antibodies to block specific individual HLA classI molecules or experiments using labeled fragments. For example,antibodies which bind selectively to HLA-A2 will prevent efficientpresentation of antigens specifically presented by HLA-A2. Thus, if thepresent peptides are presented by HLA-A2, then the inclusion ofanti-HLA-A2 antibodies in an in vitro assay will block the presentationof these antigens. An assay for determining the nature of the HLAmolecule is found in U.S. Pat. No. 5,939,526.

VII. Therapeutic Compositions/Vaccines

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations can routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term “physiologically acceptable” refers toa non-toxic material that is compatible with a biological system such asa cell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The present invention provides therapeutic compositions includingvaccine compositions comprising the CD19 or CD20 antigen peptidefragments or nucleic acids encoding these fragments as described above.Vaccines can be prepared from antigen presenting cells that have beenpulsed with the peptides or nucleic acids or immune effector cells whichhave been exposed to the peptides or nucleic acids. The vaccine cancontain a single peptide or a range of peptides that cover different orsimilar epitopes. In addition or alternatively, the vaccine can be apolyvalent vaccine where a single polypeptide can be provided withmultiple epitopes. The vaccine compositions of the present invention maybe cancer vaccines

In one embodiment of a vaccine composition, the peptide is conjugated toa carrier protein, for example a polycation (poly-L-Lysine orpoly-L-arginine), tetanus toxoid, diphtheria toxoid or oxidized KLH orthe like in order to stimulate T cell help as disclosed in U.S. Pat. No.6,344,203.

Included as part of the vaccine, substances that potentiate the immuneresponse can be administered with nucleic acid or peptide components.Such immune response potentiating compounds can be classified as eitheradjuvants or cytokines. Adjuvants can enhance the immunological responseby providing a reservoir of antigen (extracellularly or withinmacrophages), activating macrophages, and stimulating specific sets oflymphocytes. Adjuvants of many kinds are well known in the art; specificexamples include MPL (SmithKline Beecham), a congener obtained afterpurification and acid hydrolysis of Salmonella minnesota R595lipopolysaccharide, QS21 (SmithKline Beecham), a pure QA-21 saponinpurified from Quillja saponaria extract, GM-CSF, Incomplete Freund'sadjuvant, KLH and various water-in-oil emulsions prepared frombiodegradable oils such as squalene and/or tocopherol. Because of theirlymphocyte stimulatory properties, cytokines are also useful invaccination protocols. Many cytokines useful for such purposes will beknown to one of ordinary skill in the art and may include interleukin-12(IL-12) which has been shown to enhance the protective effects ofvaccines (Hall, Science, 268:1432-1434 (1995)).

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. Following immunization protocols standard in the art, initialdoses can be followed by booster doses. The administration may, forexample, be oral, intravenous, intraperitoneal, intramuscular,intracavity, subcutaneous, or transdermal. When antibodies are usedtherapeutically, a preferred route of administration is by pulmonaryaerosol. Techniques for preparing aerosol delivery systems containingantibodies are well known to those of skill in the art. Generally, suchsystems should utilize components which will not significantly impairthe biological properties of the antibodies, such as the paratopebinding capacity (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, Gennaro et al., 18 ed.: 1694-1712(1990)). Those of skill in the art can readily determine the variousparameters and conditions for producing antibody aerosols without resortto undue experimentation. When using antisense preparations of theinvention, slow intravenous administration is preferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Examples of non-aqueous solventsinclude propylene glycol, polyethylene glycol, vegetable oils such asolive oil, and injectable organic esters such as ethyl oleate.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils. If thepreparation is administered intravenously, intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like may also be present.

In cases where cells are used as a vaccine, these can be cellstransfected with coding sequences for one or both of the componentsnecessary to provoke a CTL response, or be cells that already expressboth molecules without the need for transfection. Vaccines alsoencompass naked DNA or RNA, encoding the present peptides, which can beproduced in vitro and administered via injection, particle bombardment,nasal aspiration and using other methods. Vaccines of the “naked nucleicacid” type have been demonstrated to provoke an immunological responseincluding generation of CTLs specific for the peptide encoded by thenaked nucleic acid (Ulmer et al., Science, 259:1745-1748 (1993)). When“disorder” is used herein, it refers to any pathological condition wherethe tumor rejection antigen precursor is expressed. In particularexamples of such disorders are cancers, leukemias and lymphomas.

The peptides of the present invention can also be used to elicit orenhance an immune response to an antigen encoded by a DNA vaccine. DNAvaccines may encode one or more immunostimulating antigens such that theantigen is generated in situ. For instance, the DNA vaccine can encode atumor antigen and, optionally, a peptide as described herein. In suchvaccines, the DNA can be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid expression systems, such as bacteria and viral expression systems.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter).Bacterial delivery systems involve the administration of a bacterium(such as Bacillus-Calmette-Guerrin) that expresses an epitope of aleukemia cell antigen on its cell surface. The DNA can be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. A variety ofsuitable delivery systems are disclosed, for example, in Fisher-Hoch etal., Proc Natl Acad Sci, 86:317-321 (1989); Flexner et al., Ann NY AcadSci, 569:86-103 (1989); Flexner et al., Vaccine, 8:17-21 (1990); U.S.Pat. Nos. 4,603,112; 4,769,330; 4,777,127; and 5,017,487; WO 89/01973;GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques,6:616-627 (1988); Rosenfeld et al., Science 252:431-434 (1991); Kolls etal., Proc Natl Acad Sci, 91:215-219 (1994); Kass-Eisler et al., ProcNatl Acad Sci 90:11498-11502 (1993); Guzman et al., circ. 88:2838-2848(1993); and Guzman et al., Cir Res, 73:1202-1207 (1993). Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA can also be “naked,” as described,for example, in published PCT Appl. No. WO 90/11092; and Ulmer et al.,Science, 259:1745-1749 (1993), reviewed by Cohen, Science, 259:1691-1692(1993). The uptake of naked DNA can be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.

VIII. Method of Treatment

The present invention provides a method of treating individualssuffering from leukemia. In such methods, the introduction of peptides,nucleic acids, protein binding agents, antigen presenting cells and/orimmune effector cells as described above serves as an immunotherapeutic,directing and promoting the immune system of the individual to combatleukemic cells that display the CD19 or CD20 antigen fragments. Themethods can comprise administering, through the means described above,an effective amount of any of the above compounds to a patient in needof such treatment. The methods can further comprise a course ofchemotherapy, such as with 5-FU or cisplatin, prior to administration ofthe above compounds. See, e.g., Tanaka et al., Int J Cancer, 101:265(2002).

Individuals at risk of developing cancers displaying CD19 and/or CD20,such as those having a genetic predisposition, can be treated with theformulations of the present invention in a prophylactic attempt to delayor eliminate the onset of the leukemic state. Those individuals who havealready developed cancer and who have been treated to remove the canceror are otherwise in remission are particularly susceptible to relapseand reoccurrence. As part of a treatment regimen, to combat a recurrencesuch individuals can be immunized against the cancer that they have beendiagnosed as having had. Thus, once it is known that an individual hashad a type of cancer and is at risk of a relapse, they can be immunizedin order to prepare their immune system to combat any future appearanceof the cancer.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, which potentially leads to lysisof leukemia cells. One such therapeutic approach is the administrationof autologous CTLs specific to the complex to a subject with abnormalcells of the phenotype at issue. It is within the skill of the artisanto develop such autologous CTLs in vitro. Specific production of a CTLis well known to one of ordinary skill in the art. Generally, a sampleof cells taken from a subject, such as blood cells, are contacted with acell presenting the complex and capable of provoking CTLs toproliferate. The target cell can be a transfectant. These transfectantspresent the desired complex on their surface and, when combined with aCTL of interest, stimulate the CTL's proliferation. The clonallyexpanded autologous CTLs can then be administered to the subject.

In one therapeutic methodology, referred to as adoptive transfer(Greenberg J, Immunol, 136(5):1917 (1986); Riddel et al., Science,257:238 (1992); Lynch et al, Eur J Immunol, 21:1403-1410 (1991); Kast etal., Cell, 59:603-614 (1989)), cells presenting the desired complex arecombined with CTLs thereby leading to proliferation of the CTLsspecific. The proliferated CTLs are then administered to a subject witha cellular abnormality to the complex that may be characterized bycertain of the abnormal cells presenting the particular complex. TheCTLs can then lyse the abnormal cells, thereby achieving the desiredtherapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA complex. This can be determinedeasily, as the art is familiar with methods for identifying cellspresenting a particular HLA molecule. The art also encompasses methodsof identifying cells expressing DNA of the pertinent sequences, in thiscase a leukemia associated gene sequence. Once cells presenting therelevant complex are identified via the foregoing screening methodology,they can be combined with a sample from a patient, where the samplecontains CTLs. If the complex presenting cells are lysed by the mixedCTL sample, then it can be assumed that a leukemia associated gene isbeing presented, and the subject is an appropriate candidate for thetherapeutic approaches set forth herein.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo using anumber of approaches. One approach is the use of non-proliferative cellsexpressing the relevant HLA complex, such as antigen presenting cells.The cells used in this approach can be those that normally express thecomplex, such as irradiated tumor cells or cells they can be transfectedwith one or both of the genes necessary for presentation of the complex.Chen et al., Proc Natl Acad Sci USA, 88:110-114 (1991), whichexemplifies the transfected cell approach, shows the use of transfectedcells expressing HPV E7 peptides in a therapeutic regime. In differentembodiments, various cell types that express the complex can be used.Vectors carrying one or both of CD19 and/or CD20 can be used. Viral orbacterial vectors are especially preferred. In certain embodiments, thenucleic acid can be incorporated into an expression vector. Expressionvectors can be unmodified extrachromosomal nucleic acids, plasmids orviral genomes constructed or modified to enable insertion of exogenousnucleic acids, such as those encoding the present peptides. Nucleicacids encoding these peptides can also be inserted into a retroviralgenome, thereby facilitating integration of the nucleic acid into thegenome of the target tissue or cell type. In these systems, the gene ofinterest is “carried” by carrier, e.g., a Vaccinia virus, retrovirus orthe bacteria BCG, and the materials defacto “infect” host cells. Thecells that result from this “infection” present the complex of interest,and are recognized by autologous CTLs, which then proliferate.

IX. Kits

The invention also provides isolated proteins and peptides, andantibodies to those proteins and peptides. Kits containing any of theforegoing molecules, alone or in combination, are additionally provided.The foregoing kits can be used in the diagnosis or treatment ofconditions characterized by the expression of the present peptides. Thekits can also be used to pulse antigen presenting cells ort-lymphocytes, and, as such, can contain appropriate culture media,culture media supplements such as cytokines, disposable laboratoryequipment and the like. Examples of such kit components can be found inthe following examples.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products. The notice may reflect approval by the agency ofmanufacture, use, or sale for human administration. In addition, thepharmaceutical compositions can be employed in conjunction with othertherapeutic compounds.

The invention in another aspect involves a kit for detecting thepresence of the expression of the present polypeptide. Such kits employtwo or more of the above-described nucleic acid molecules isolated inseparate containers and packaged in a single package. In one such kit, apair of isolated nucleic acid molecules is provided. In certainembodiments, the pair of isolated nucleic acid molecules are PCRprimers.

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components can be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

A total of 556 amino acids of CD19 protein, listed above as SEQ ID NO:13, were retrieved from the SWISS-PROT databank and analyzed forHLA-A2.1 binding epitopes as nonamers by the peptide motif searchsoftware SYFPEITHI which is supported by DFG-Sonderforschungsbereich 510and the European Union: EU BIOMED CT95-1627, BIOTECH CT95-0263, and EUQLQ-CT-1999-00713. The algorithms used are based on the book MHC Ligandsand Peptide Motifs, by H. G. Rammensee et al., Landes Bioscience, 1997.The prediction of the SYFPEITHI program is based on published motifs(pool sequencing, natural ligands) and the score is calculated by theconsideration of specific amino acids of the peptide in a numeric valuedepending on whether the peptide is a carrying anchor, auxiliary anchor,or preferred residue. Ideal anchors are given 10 points, unusual anchors6-8 points, auxiliary anchors 4-6 points and preferred residues 1-4points. Amino acids that are regarded as having a negative effect on thebinding are given values between −1 and −3. Besides a prediction forbinding, the preferred amino acids in the peptide of CD19 antigen wereexamined for the possible immunogenic epitopes. Peptides are groupedbased on overall score. As shown, the values for the tested CD19peptides ranged from an overall high score of 29 to an overall low scoreof 10.

Position 1 2 3 4 5 6 7 8 9 Score SEQ ID NO. 63 K L S L G L P G L 29 32299 Y L I F C L C S L 29 33 5 R L L F F L L F L 27 1 284 L L R T G G W KV 26 34 296 T L A Y L I F C L 26 2 353 S L P T P T S G L 26 35 10 L L FL T P M E V 25 3 303 C L C S L V G I L 25 36 508 Y A A P Q L H S I 25 37150 K L M S P K L Y V 24 4 310 I L H L Q R A L V 24 38 65 S L G L P G LG I 23 39 128 D L G G L G C G L 23 40 309 G I L H L Q R A L 23 41 245 GL L L P R A T A 22 42 300 L I F C L C S L V 22 43 6 L L F F L L F L T 2144 70 G L G I H M R P L 21 45 74 H M R P L A S W L 21 46 279 V L W H W LL R T 21 47 505 G I L Y A A P Q L 21 48 27 K V E E G D N A V 20 49 183 SL S Q D L T M A 20 50 188 L T M A P G S T L 20 51 194 S T L W L S C G V20 52 346 N Q Y G N V L S L 20 53 105 K A W Q P G W T V 19 54 190 M A PG S T L W L 19 55 238 D M W V M E T G L 19 56 266 L T M S F H L E I 1957 271 H L E I T A R P V 19 58 292 V S A V T L A Y L 19 59 305 C S L V GI L H L 19 60 306 S L V G I L H L Q 19 61 502 D M R G I L Y A A 19 62 56S P L K P F L K L 18 63 67 G L P G L G I H M 18 64 79 A S W L F I F N V18 65 151 L M S P K L Y V W 18 66 220 K G P K S L L S L 18 67 224 S L LS L E L K D 18 68 240 W V M E T G L L L 18 69 302 F C L C S L V G I 1870 20 P E E P L V V K V 17 71 50 L T W S R E S P L 17 72 113 V N V E G SG E L 17 73 179 S L N Q S L S Q D 17 74 197 W L S C G V P P D 17 75 246L L L P R A T A Q 17 76 351 V L S L P T P T S 17 77 363 R A Q R W A A GL 17 78 467 E L T Q P V A R T 17 79 9 F L L F L T P M E 16 80 31 G D N AV L Q C L 16 81 34 A V L Q C L K G T 16 82 81 W L F I F N V S Q 16 83107 W Q P G W T V N V 16 84 121 L F R W N V S D L 16 85 131 G L G C G LK N R 16 86 247 L L P R A T A Q D 16 87 287 T G G W K V S A V 16 88 293S A V T L A Y L I 16 89 312 H L Q R A L V L R 16 90 370 G L G G T A P SY 16 91 42 T S D G P T Q Q L 15 92 58 L K P F L K L S L 15 93 124 W N VS D L G G L 15 94 199 S C G V P P D S V 15 95 225 L L S L E L K D D 1596 241 V M E T G L L L P 15 97 267 T M S F H L E I T 15 98 272 L E I T AR P V L 15 99 311 L H L Q R A L V L 15 100 318 V L R R K R K R M 15 101360 G L G R A Q R W A 15 102 367 W A A G L G G T A 15 103 439 Y E N P ED E P L 15 104 484 S A W D P S R E A 15 105 494 S L G S Q S Y E D 15 10612 F L T P M E V R P 14 107 17 E V R P E E P L V 14 108 18 V R P E E P LV V 14 109 23 P L V V K V E E G 14 110 28 V E E G D N A V L 14 111 35 VL Q C L K G T S 14 112 54 R E S P L K P F L 14 113 61 F L K L S L G L P14 114 83 F I F N V S Q Q M 14 115 88 S Q Q M G G F Y L 14 116 95 Y L CQ P G P P S 14 117 143 G P S S P S G K L 14 118 148 S G K L M S P K L 14119 158 V W A K D R P E I 14 120 166 I W E G E P P C V 14 121 187 D L TM A P G S T 14 122 189 T M A P G S T L W 14 123 217 V H P K G P K S L 14124 274 I T A R P V L W H 14 125 276 A R P V L W H W L 14 126 286 R T GG W K V S A 14 127 289 G W K V S A V T L 14 128 391 L G S R S P P G V 14129 468 L T Q P V A R T M 14 130 478 F L S P H G S A W 14 131 77 P L A SW L F I F 13 132 180 L N Q S L S Q D L 13 133 264 G N L T M S F H L 13134 366 R W A A G L G G T 13 135 377 S Y G N P S S D V 13 136 383 S D VQ A D G A L 13 137 390 A L G S R S P P G 13 138 426 N L G Q D Q L S Q 13139 460 S Y E N E D E E L 13 140 471 P V A R T M D F L 13 141 506 I L YA A P Q L H 13 142 2 P P P R L L F F L 12 143 8 F F L L F L T P M 12 14438 C L K G T S D G P 12 145 57 P L K P F L K L S 12 146 60 P F L K L S LG L 12 147 66 L G L P G L G I H 12 148 73 I H M R P L A S W 12 148 90 QM G G F Y L C Q 12 149 118 S G E L F R W N V 12 150 120 E L F R W N V SD 12 151 127 S D L G G L G C G 12 152 135 G L K N R S S E G 12 153 172 PC V P P R D S L 12 154 207 V S R G P L S W T 12 155 218 H P K G P K S LL 12 156 227 S L E L K D D R P 12 157 236 A R D M W V M E T 12 158 239 MW V M E T G L L 12 159 258 K Y Y C H R G N L 12 160 283 W L L R T G G WK 12 161 295 V T L A Y L I F C 12 162 369 A G L G G T A P S 12 163 386 QA D G A L G S R 12 164 424 D S N L G Q D Q L 12 165 498 Q S Y E D M R GI 12 166 499 S Y E D M R G I L 12 167 515 S I R G Q P G P N 12 168 16 ME V R P E E P L 11 169 49 Q L T W S R E S P 11 170 72 G I H M R P L A S11 171 76 R P L A S W L F I 11 172 146 S P S G K L M S P 11 173 155 K LY V W A K D R 11 174 165 E I W E G E P P C 11 175 195 T L W L S C G V P11 176 201 G V P P D S V S R 11 177 206 S V S R G P L S W 11 178 222 P KS L L S L E L 11 179 243 E T G L L L P R A 11 180 260 Y C H R G N L T M11 181 308 V G I L H L Q R A 11 182 316 A L V L R R K R K 11 183 325 R MT D P T R R F 11 184 343 G P Q N Q Y G N V 11 185 350 N V L S L P T P T11 186 431 Q L S Q D G S G Y 11 187 464 E D E E L T Q P V 11 188 474 R TM D F L S P H 11 189 487 D P S R E A T S L 11 190 547 G G R M G T W S T11 191 13 L T P M E V R P E 10 192 176 P R D S L N Q S L 10 193 209 R GP L S W T H V 10 194 216 H V H P K G P K S 10 195 251 A T A Q D A G K Y10 196 265 N L T M S F H L E 10 197 275 T A R P V L W H W 10 198 277 R PV L W H W L L 10 199 317 L V L R R K R K R 10 200 328 D P T R R F F K V10 201 344 P Q N Q Y G N V L 10 202 419 E F Y E N D S N L 10 203 492 A TS L G S Q S Y 10 204 512 Q L H S I R G Q P 10 205 542 P A W G G G G R M10 206

A similar search for CD20 provided the following sequences:

Position 1 2 3 4 5 6 7 8 9 Score SEQ ID NO. 188 S L F L G I L S V 32 6127 A I S G M I L S I 27 7 64 I A L G G L L M I 25 205 68 G L L M I P AG I 24 206 87 P L W G G I M Y I 24 207 116 K M I M N S L S L 24 208 190F L G I L S V M L 24 209 198 L I F A F F Q E L 24 210 56 Q I M N G L F HI 23 211 92 I M Y I I S G S L 23 212 130 G M I L S I M D I 23 234 241 EI K E E V V G L 23 214 95 I I S G S L L A A 22 215 123 S L F A A I S G M22 216 22 A M Q S G P K P L 21 217 135 I M D I L N I K I 21 218 185 S IQ S L F L G I 21 219 29 P L F R R M S S L 20 220 76 I Y A P I C V T V 20221 131 M I L S I M D I L 20 222 154 F I R A H T P Y I 20 8 193 I L S VM L I F A 20 223 238 Q T I E I K E E V 20 224 36 S L V G P T Q S F 19225 57 I M N G L F H I A 19 226 65 A L G G L L M I P 19 227 75 G I Y A PI C V T 19 228 94 Y I I S G S L L A 19 229 70 L M I P A G I Y A 18 23099 S L L A A T E K N 18 231 138 I L N I K I S H F 18 232 147 L K M E S LN F I 18 10 151 S L N F I R A H T 18 9 206 L V I A G I V E N 18 233 239T I E I K E E V V 18 234 33 R M S S L V G P T 17 235 50 K T L G A V Q IM 17 236 53 G A V Q I M N G L 17 237 61 L F H I A L G G L 17 238 63 H IA L G G L L M 17 239 118 I M N S L S L F A 17 240 125 F A A I S G M I L17 241 133 L S I M D I L N I 17 242 134 S I M D I L N I K 17 243 186 I QS L F L G I L 17 244 207 V I A G I V E N E 17 245 71 M I P A G I Y A P16 246 72 I P A G I Y A P I 16 247 111 C L V K G K M I M 16 248 144 S HF L K M E S L 16 11 191 L G I L S V M L I 16 249 47 R E S K T L G A V 15250 121 S L S L F A A I S 15 251 139 L N I K I S H F L 15 252 181 Q Y CY S I Q S L 15 253 203 F Q E L V I A G I 15 254 222 R P K S N I V L L 15255 228 V L L S A E E K K 15 256 60 G L F H I A L G G 14 257 62 F H I AL G G L L 14 258 69 L L M I P A G I Y 14 259 84 V W Y P L W G G I 14 26091 G I M Y I I S G S 14 261 96 I S G S L L A A T 14 262 120 N S L S L FA A I 14 263 161 Y I N I Y N C E P 14 264 189 L F L G I L S V M 14 265199 I F A F F Q E L V 14 266 200 F A F F Q E L V I 14 267 220 C S R P KS N I V 14 268 257 K N E E D I E I I 14 269 16 P M K G P I A M Q 13 27044 F F M R E S K T L 13 271 49 S K T L G A V Q I 13 272 74 A G I Y A P IC V 13 273 80 I C V T V W Y P L 13 274 88 L W G G I M Y I I 13 275 100 LL A A T E K N S 13 276 114 K G K M I M N S L 13 277 117 M I M N S L S LF 13 278 142 K I S H F L K M E 13 279 156 R A H T P Y I N I 13 280 201 AF F Q E L V I A 13 281 210 G I V E N E W K R 13 282 224 K S N I V L L SA 13 283 226 N I V L L S A E E 13 284 229 L L S A E E K K E 13 285 30 LF R R M S S L V 12 286 58 M N G L F H I A L 12 287 93 M Y I I S G S L L12 288 124 L F A A I S G M I 12 289 132 I L S I M D I L N 12 290 192 G IL S V M L I F 12 291 196 V M L I F A F F Q 12 292 204 Q E L V I A G I V12 293 221 S R P K S N I V L 12 294 231 S A E E K K E Q T 12 295 232 A EE K K E Q T I 12 296 248 G L T E T S S Q P 12 297 264 I I P I Q E E E E12 298 7 S V N G T F P A E 11 299 45 F M R E S K T L G 11 300 101 L A AT E K N S R 11 301 110 K C L V K G K M I 11 302 137 D I L N I K I S H 11303 141 I K I S H F L K M 11 304 146 F L K M E S L N F 11 305 163 N I YN C E P A N 11 306 183 C Y S I Q S L F L 11 307 197 M L I F A F F Q E 11308 242 I K E E V V G L T 11 309 262 I E I I P I Q E E 11 310 263 E I IP I Q E E E 11 311 3 T P R N S V N G T 10 312 13 P A E P M K G P I 10313 21 I A M Q S G P K P 10 314 51 T L G A V Q I M N 10 315 77 Y A P I CV T V W 10 316 83 T V W Y P L W G G 10 317 90 G G I M Y I I S G 10 318126 A A I S G M I L S 10 319 128 I S G M I L S I M 10 320 225 S N I V LL S A E 10 321 227 I V L L S A E E K 10 322 234 E K K E Q T I E I 10 323254 S Q P K N E E D I 10 324 256 P K N E E D I E I 10 325

As shown, the values for the tested CD20 peptides ranged from an overallhigh score of 32 to an overall low score of 10.

Based on high HLA-A2.1 binding scores, several 9 mer fragments of theCD19 peptide (SEQ ID NO: 13) or CD20 peptide (SEQ ID NO: 14) wereidentified using SYFPEITHI software and are listed above. The scoredemonstrates the calculated potential capability for binding to a HLA-A2molecule.

The following peptides were tested for their ability to bind toHLA-A2.1+T2 cells and their binding scores under Brefeldin A treatmentwere evaluated. Tourdot at al., Eur J Immunol, 30:3411-3421 (2000). Thisexperiment was performed to measure the peptide/HLA-A2.1 complexstability using the native or modified CD19 or CD20 peptides. As theimmunogenicity of a peptide depends primarily on its capacity tostabilize the HLA-A2.1 molecules, a peptide that greatly stabilizesHLA-A2.1 could generate more effective cytotoxic T cells that recognizeleukemia cells.

T2 cells, a transporter antigen processing (TAP) gene-deficient cellline that express only HLA-A2.1 MHC class I molecules (Zweerink H J etal., J Immunol, 150:1763-1771 (1993)), were used to evaluate CD19 orCD20 peptide specific binding to HLA-A2.1. HLA-A2.1-specific influenzavirus matrix peptide 58-66 (GILGFVFTL, SEQ ID NO: 31), which is known tobind very tightly to HLA-A2.1, was used as an HLA-A2.1-specific controlpeptide.

Peptide Synthesis

The peptides set forth in the table below were synthesized by standardfmoc (9-fluorenylmethyl-oxycarbonyl) chemistries and were purifiedto >90% using reverse-phase chromatography (Biosynthesis, Lewisville,Tex.). A HLA-A2.1-specific influenza virus protein matrix peptide(GILGFVFTL: residues 58-66, SEQ ID NO: 31) was synthesized and used as apositive control in these studies. The identity of each peptide wasvalidated by measuring mass-spectrometry for molecular weight.

Brefeldin A treatment Peptide Sequence None 0 hr 2 hr 4 hr 6 hrOvernight T2 alone 215 226 200 187 157 255 T2 plus influenza viruspeptide SEQ ID NO: 31 GILGFVFTL 605 807 897 909 759 504 CD19 SEQ ID NO:1 RLLFFLLFL N/A 167 202 174 152 248 SEQ ID NO: 2 TLAYLIFCL 363 530 608513 387 312 SEQ ID NO: 3 LLFLTPMEV 556 770 924 880 573 453 SEQ ID NO: 4KLMSPKLYV 600 683 759 796 642 540 SEQ ID NO: 5 LLFFLLFLV 164 200 202 163143 200 CD20 SEQ ID NO: 6 SLFLGILSV 637 672 703 762 770 410 SEQ ID NO: 7AISGMILSI 162 220 256 165 144 232 SEQ ID NO: 8 FIRAHTPYI 167 232 215 192154 250 SEQ ID NO: 9 SLNFIRAHT 172 212 200 180 128 195 SEQ ID NO: 10LKMESLNFI 215 245 291 227 190 225 SEQ ID NO: 11 SHFLKMESL 172 216 218199 127 277

As can be seen from these results, the peptides of SEQ ID NOS: 2, 3, 4and 6 gave good and prolonged stable binding. Several other peptidesprovided good initial binding over baseline values.

Example 2

The present example illustrates that that the peptides of the inventionare capable of inducing a T-lymphocyte response. FIG. 1 is a timeline ofevents performed for the generation of CD20 peptide-specific cytotoxicT-lymphocytes. A similar timeline can be used to produce CD19peptide-specific cytotoxic T lymphocytes. CD19 or CD20 peptide-specificcytotoxic T lymphocytes were generated by stimulating T lymphocytes fromHLA-A2+ normal donors with dendritic cells pulsed with a peptide ofinterest. In this and the following examples, antigen presenting cellswere pulsed at 150 micrograms of the peptide per 1 million antigenpresenting cells with SLFLGILSV (SEQ ID NO: 6). CD19 peptide specificcytotoxic T-lymphocytes were generated by contacting TLAYLIFCL (SEQ IDNO: 2), KLMSPKLYV (SEQ ID NO: 4), or LLFLTPMEV (SEQ ID NO: 3) with thecytotoxic T-lymphocytes once per week for 4 to 5 weeks.

Other peptides that include the sequences listed above can be similarlytested by the skilled artisan in the course of normal experimentation todetermine whether peptides including any of the recited sequences can beeffectively used as described herein.

The antigen presenting cells were stimulated one to four times bycontacting them with effector cells at different effector:target cellratios. The effector cells were then contacted with HLA-A2.1 positiveST486 cells and the cytotoxicity to the ST486 cells were measured. ST486cells are Burkett's lymphoma cells which are HLA-A2.1 positive. Theratios of target: effectorized were 1:1, 10:1, 20:1, and 60:1. Theresults for CD19 peptide specific cytotoxic T-lymphocytes are set forthin FIG. 2 a. The results for CD20 peptide specific cytotoxicT-lymphocytes are shown in FIG. 2 b and FIG. 3. FIG. 4 shows theexpansion of CD20 peptide specific cytotoxic T-lymphocytes over timeusing CD3/CD28 beads and IL-2. FIG. 5 shows the cytotoxic activity ofexpanded CD20 peptide specific cytotoxic T-lymphocytes to ST486 atdifferent effector:target cell ratios. FIGS. demonstrate that theexpanded cytotoxic T-lymphocytes retain their cytotoxic activity afterexpansion. CTLs were expanded in AIM-V media containing CD3/CD28 beadsto stimulate T cells and IL-2 to proliferate CTLs at cell density of0.5-0.0×10⁶ cells/ml.

Cell Lines

A ST486 cell line, purchased from American Tissue Culture Collection(ATCC) was maintained in liquid culture in RPMI 1640 and 10% fetal calfserum (FCS; Biowhittaker, Walkersville, Md.). T2 cells, a human B and Tcell hybrid expressing HLA-A2.1, were maintained in RPMI 1640 plus 20%FCS and used as antigen presenting cells in these studies.

MHC Peptide Binding Assay

The assay for peptide binding to HLA-A2.1 was performed (Nijman H W etal., Eur J Immunol, 23:1215-1219 (1993)) using the TAP-deficient T2hybrid cell line, which is known to up-regulate HLA-A2.1 expression onthe cell surface by acquiring only exogenous epitope (Salter et al. EMBOJ, 5:943-949 (1986); Zweerink H J et al., J Immunol, 150:1763-1771(1993)). T2 cells were washed and resuspended in serum-free AIM-V™(Gibco-Life Technologies, Rockville, Md.) at a final concentration of1×10⁶ cells/ml and transferred into a 24-well tissue culture plate.Cells were pulsed with respective CD19 or CD20 peptides at differentconcentrations (5-150 μg/ml) or HLA-A2.1-specific influenza virusprotein matrix peptide (30 μg/ml) plus 3 μg human P2-microglobulin(Sigma), and incubated for overnight at 37° C., 5% CO₂ in humidifiedair. After incubation, cells were washed once with PBS containing 3%FCS, and stained with mouse anti-HLA-A2.1 monoclonal antibody for 15minutes at 4° C. After washing, the cells were incubated with goatanti-mouse IgG(F(ab′)2)-FITC for 15 minutes at 4° C. The cells werewashed once, and fluorescence was measured on a FACSort™ flow cytometer(Becton Dickson, San Jose, Calif.). The fluorescence index wascalculated as follows: (mean channel fluorescence of sample—mean channelfluorescence of unstained control cells)/mean channel fluorescence ofunstained control cells.

Cell Isolation

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized whole blood of healthy HLA-A2.1+ donors by standard gradientcentrifugation with Ficoll-Paque™ Plus (Amersham Pharmacia Biotech AB,Uppsala, Sweden). PBMCs were harvested from the interface, washed twice,and resuspended in PBS supplemented with 5 mM EDTA and 0.5% human serumalbumin. Informed consent was obtained from all donors and the protocolwas approved by the Rush Medical School Institutional Review Board.

CD14+ monocytes were separated from the isolated PBMCs using a magneticsorting technique (Miltenyi Biotec, Auburn, Calif.). PBMCs wereincubated with colloidal super-paramagnetic microbeads conjugated withanti-human CD14 mAb for 15 minutes at 4° C., and passed over a column ina magnetic field. After washing, positively enriched CD14+ cells wereeluted from the magnetic columns. Purity (mean±standard deviation) ofCD14+ monocytes was examined by flow cytometry and was found to be92±4%.

CD3+ T cells were isolated from the monocyte depleted cell fractionsusing the Pan T cell isolation kit from Miltenyi Biotec (Auburn,Calif.). The T cell isolation was done by depletion of B cells, NKcells, early erythroid cells, platelets and basophils by indirectlylabeling with a cocktail of hapten-conjugated CD11b, CD16, CD19, CD36and CD56 antibodies, and MACS® microbeads (Miltenyi Biotech) coupled toan anti-hapten monoclonal antibody. The effluent (negative fractioncells) was collected from the column as the enriched CD3+ T cellfraction. Purity (mean±standard deviation) of CD3+ T cells was examinedby flow cytometry and was found to be 94±4%.

Dendritic Cell (DC) Generation.

Immature DCs can be generated according to modified protocols of RomaniN, et al., J Exp Med, 180:83-89 (1994); and Bakker et al., Cancer Res,55:5330-5334 (1995). Briefly, fresh or frozen/thawed CD14+ cells arecultured in RPMI 1640 medium (Gibco-Life Technologies, Gaithersburg,Md.) supplemented with 10% FCS, 1,000 U/ml GM-CSF and 1,000 U/ml IL-4.The cell cultures are fed with fresh medium and GM-CSF and IL-4 everyother day and cell differentiation is monitored by light microscopy. Onday 7, the cultures are supplemented with different combinations of DCmaturation factors such as lipopolysaccharide (100 U/ml), TNF-α (10ng/ml), or IFN-α (1,000 U/ml) plus TNF-α (10 ng/ml). After three days ofincubation, mature DCs (mDC) are harvested and their phenotypes areevaluated by flow cytometry. The maturation factor(s) yielding optimalDC maturation are determined and used to generate mDCs for peptidepulsing in upcoming studies.

Induction of Peptide-Specific CTLs.

Two different types of antigen-presenting cells (APCs), mDCs and T2cells, are used to generate CD19 or CD20 peptide-specific CTLs. APCs arewashed three times in serum-free AIM-V™ culture media and pulsed withpeptide at 150 μg/ml in the media overnight. The peptide-loaded APCs arethen irradiated at 10 Gy, washed once, and resuspended in RPMI 1640media supplemented with 10% human AB serum (Biowhittaker, Walkersville,Md.). Peptide-pulsed APCs are used to prime autologous CD3+ T cells at a1:20 stimulator-to-responder cell ratio in RPMI 1640 media supplementedwith 10% human AB serum, 5 ng/ml IL-6, 20 ng/ml IL-7, and 1 ng/ml IL-12.CTL cultures are restimulated weekly for a total of 4 cycles ofstimulation. IL-2 (50 U/ml) is added to the culture one day after thethird stimulation and the cells are fed three times a week with freshmedium containing the cytokines.

Cytotoxicity Assay

The cytolytic activity of the CD19 or CD20 peptide-specific CTLs ismeasured in a standard ⁵¹Cr-release assay. The CTLs (effector cells) areseeded with ⁵¹Cr-labeled 5×10³ lymphoma cells (target cells) per well atvarious effector:target cell ratios in 96-well U-bottom microtiterplates and incubated for 4 hours at 37° C., 5% CO₂. After theincubation, the supernatants (100 ul) are harvested and the specific⁵¹Cr-release is measured using a Beckman LS6500 liquid scintillationcounter (Beckman Coulter, Brea, Calif.). The percent specific cell lysisis calculated as [(experimental release−spontaneous release)÷(maximumrelease−spontaneous release)]. Maximum release is determined fromdetergent-releasable target cell counts and spontaneous release isdetermined from the target cell counts in the absence of CTLs.

Cold Target Inhibition Assays

Antigen-specific lysis is evaluated in a cold target inhibition assay byanalyzing the capacity of unlabeled lymphoma cells to block lysis of⁵¹Cr-labeled lymphoma cells. Effector cells are incubated with an equalnumber of the unlabeled “cold” target cells (ML-2) for 1 hour at 37° C.,5% CO₂ before the addition of ⁵¹Cr-labeled “hot” lymphoma target cells.Although ML-2 cells, a human, peripheral blood, acute myelomonocyticleukemia cell line are used in certain experiments of the invention, theskilled artisan understands that alternative cell lines may be used withthe invention. After a 4-hour incubation, the supernatants (100 ul) areharvested and the specific ⁵¹Cr-release is measured. The inhibition oflymphoma-specific lysis is measured by comparing the percentcytotoxicity of the effector cells incubated with or without theunlabeled “cold” target cells.

IFN-γ Release by CD19 or CD20 Peptide-Specific CTLs

IFN-γ release by the CTLs is measured using an IFN-γ ELISA kit(PBL-Biomedical Lab., Piscataway, N.J.). Briefly, IFN-γ standards or thesupernatant from CD19 or CD20 peptide-specific CTL cultures aretransferred into a 96-well plate pre-coated with anti-human IFN-γcapture monoclonal antibody and incubated for 1 hour in a closed chamberat 24° C. After washing the plate with PBS/0.05% Tween 20, anti-humanIFN-γ antibody is added to the wells and incubated for 1 hour at 24° C.Wells are then developed by incubation with horseradish peroxidaseconjugate and TMB substrate solution. Stop solution is added to eachwell and the absorbance is determined at 450 nm with a SpectraMAX Plusplate reader (Stratagene, La Jolla, Calif.). The amount of cytokinepresent in the CTL culture supernatant is calculated based on the IFN-γstandard curve.

Phenotypic Analysis of CD19 or CD20 Peptide-Specific CTLs

CTLs are stained with anti-CD8-FITC, —CD45RA-FITC or —CD28-PE,—CD45RO-PE, or —CD69-PE monoclonal antibodies for 15 minutes at 4° C.After incubation, the cells are washed and analyzed by flow cytometry.Live gating of the forward and scatter channels is used to excludedebris and to selectively acquire the lymphocyte population foranalysis. Individual fluorescence data are determined using CellQuest™v2.1 acquisition and analysis software (Becton Dickinson, FranklinLakes, N.J.).

Peptide-MHC Tetramer Staining

A Streptavidin-PE-labeled HLA-2.1/peptide tetramer is produced, usingeither provided peptide or provided sequence, to Beckman Coulter(Fullerton, Calif.) using the methods described by Altman J D et al.,Science, 274:94-96 (1996). Two-color flow cytometry assays are performedby stainings with anti-CD8-FITC and tetramer-PE. Briefly, the CTLs(2×10⁵ cells) are stained with 300 ng of tetramer and incubated for 30minutes at 37° C. After a washing, the cells are stained withanti-CD8-FITC mAb for 15 minutes at 4° C. Cells are washed and analyzedby flow cytometry.

Results Identification of HLA-A2.1-Specific CD19 or CD20 Epitope

The results, expressed as the Fluorescence Index (HLA-A2.1 mean channelfluorescence T2 cells pulsed with β2 microglobulin and CD19 or CD20peptide÷HLA-A2.1 mean channel fluorescence T2 cells pulsed with β2microglobulin) are used to select the best HLA-A2.1 binding peptide. AFluorescence Index (FI) of >1.0 indicates the up-regulation of HLA-A2.1molecules by peptide binding on the surface of T2 cells. Based on theseresults, the specific peptides are chosen for evaluation as a potentialimmunogenic epitope for use in generating peptide-specific CTLs againsttarget cells.

Dendritic and T2 Cells as Antigen Presenting Cells

In this example, immature DCs obtained by the culture of CD14+ monocyteswith GM-CSF (1,000 U/ml) and IL-4 (1,000 U/ml) are induced to undergomaturation by incubation with LPS (100 Units/ml), TNF-α (10 ng/ml) orTNF-α (10 ng/ml)+IFN-γ (50 ng/ml) during the final three days of theculture period. Flow cytometric analysis of the respective DC culturesshows a phenotypic profile characterized by high expression of CD40,CD80, CD83, and/or CD86. HLA-DR and no expression of CD3 or CD14 (datanot shown). The highest up-regulation of the co-immunostimulatory (CD80and CD86) and HLA-A2.1 MHC class I molecules is detected on DCs treatedwith TNF-α+IFN-γ (Table 5). LPS or TNF-α alone also induced the highexpression of CD80, CD86 and HLA-A2.1 molecules compared to theGM-CSF+IL-4 (immature DC) control group. However, this up-regulation wasnot as high as seen with the TNF-α+IFN-γ treated combination.

The phenotype of the T2 cell line is evaluated to determine itspotential for use as an alternative type of antigen presenting cell. Theresults (Table 4) show that T2 cells express high levels ofco-immunostimulatory and HLA-A2.1 molecules. The expression levels ofCD83 and CD86 molecules on T2 cells are comparable to those observed onmDCs (Table 4). The expression of CD80 is higher on T2 cells compared tomDCs. The phenotypic profiles of both the mDCs and T2 cells make themideal candidates for use as antigen presenting cells in the generationof CD19 or CD20 peptide-specific CTLs.

TABLE 4 Phenotypic analysis of mature dendritic (mDC) and T2antigen-presenting cells. HLA-A2.1 CD80 CD86 CD83 Immature DCs 94 56 21ND¹ DCs matured by LPS 316 71 40 ND DCs matured by TNF-α 475 107 96 NDDCs matured by IFN-γ + TNF-α 749 137 149 67 T2 cells 577 1214 155 50 ND¹= not done Phenotypic analysis of culture derived mDC or T2antigen-presenting cells. Immature DCs are generated in vitro from CD14+monocytes of HLA-A2.1+ normal donors by incubation with GM-CSF and IL-4for 10 days in liquid culture. Immature DCs are induced to undergomaturation by the addition of LPS, TNF-α or TNF-α + IFN-γ during thelast three days of culture. Results are expressed as the mean channelfluorescence (MCF) for each antigen tested. Dendritic cellsmatured withTNF-α + IFN-γ display the highest levels of HLA-A2.1, CD80, and CD86expression compared to LPS or TNF-α treated dendritic cell cultures. TheT2 cell line had the highest level of CD80 expression and similar levelsof HLA-A2.1 and CD86 expression to the TNF-α + IFN-γ dendritic cells.

Cytolytic Activity by CD19 or CD20 Peptide-Specific CTLs

CD19 or CD20 peptide-specific CTLs are generated by repeated stimulationof T-lymphocytes from healthy HLA-A2.1+ donors with peptide-pulsedautologous mDC. CTLs are harvested one week after the respective peptidestimulation and examined for their cytolytic activity against the ST486lymphoma cell line.

Autologous mDCs or T2 cells as antigen presenting cells are alsoevaluated to induce peptide-specific CTLs. T-lymphocytes from healthyHLA-A2.1+ donors are stimulated with either autologous mDC or T2 cellspulsed with the HLA-A2.1-specific peptide. The CTLs are harvested oneweek after the second, third or fourth stimulation and analyzed fortheir cytotoxic activities against the ST486 cell line. Generally,generating CTLs with a minimum of three cycles of stimulation withpeptide-pulsed mDCs to obtain highly effective CD19 or CD20peptide-specific CTLs is preferred. In further experimentation, thepossibility of the inhibition of normal cells expressing low levels ofCD19 or CD20 antigen by the CD19 or CD20 peptide-specific CTLs isexamined. CTLs are generated as previously described using mDCs or T2cells and examined a week after the first, second, third or fourthstimulation.

Lymphoma-Specific Cell Lysis

Lymphoma-specific cell lysis by CD19 or CD20 peptide-specific CTLs isconfirmed using a cold target inhibition assay. In this assay, CTLs arepre-incubation with “cold” ST486 cells for 1 hour before the addition of⁵¹Cr-labeled (“hot”) ST486 target cells. B cell lymphoma-specificcytotoxicity by CD19 or CD20 peptide-specific CTLs is confirmed usingthe cold target inhibition assay.

IFN-γ Elisa

The secretion of cytokines by antigen-specific T cells helps determinetheir effector cell function. IFN-γ secretion by antigen-specific Tcells has been shown to contribute to host defense by initiating apotent local inflammatory response. Generally, IFN-γ secretion serves asan important cytokine against tumor progression by orienting Tlymphocytes into the Th1-subtype (Ikeda H et al., Cytokine Growth FactorRev, 13:95-109 (2002); Beatty G L et al., Immunol Res, 24:201-210(2001)). The CD20 peptide-specific CTLs are evaluated for secretion ofIFN-γ as a way to analyze their potential anti-cancer activity based onthe Th1 cell subtype. Supernatants from HLA-A2.1+ peptide-specific CTLcultures are analyzed for IFN-γ production after repeated stimulationwith the peptide. The production of IFN-γ by the peptide-specific CTLcultures are shown in FIG. 6. IFN-γ production by CD19 peptide-specificCTLs can be analyzed in a similar manner.

Isotypes of CD19 or CD20 Peptide-Specific CTLs

Phenotypic analysis of the CD19 or CD20 peptide-specific CTLs isperformed to determine the expression of antigenic markers includingCD8, CD69, CD45RA, CD45RO, and CD28 on the cell surfaces. The phenotypicanalysis of CD19 or CD20 peptide specific CTLs is determined by flowcytometry following repeated stimulation of the CTLs with mDCs pulsedwith peptide.

Detection of CD19 or CD20 Peptide-Recognizing CTLs by Tetramer Staining

The CTL population recognizing specific peptides is characterized usingpeptide specific-HLA-A2.1-tetramers. Peptide-HLA-A2.1 tetramers arecomplexes of four HLA-A2.1 molecules associated with a specific peptideand a fluorochrome (Altman J D et al., Science, 274:94-96 (1996)). Thecomplexes bind to a distinct set of T cell receptors on a subset of CD8+T cells that recognize the specific peptide. CTLs recognizing thepeptides are identified by staining with peptide-HLA-A2.1-tetramers-PEand CD8-FITC antibodies.

Determination of CD19 or CD20 Epitope Specificity to HLA

In this example, the CD19 or CD20 epitope specific to HLA-A2.1 isexamined. This particular epitope was examined as this is the mostdominant HLA class I molecule, representing approximately 50% of NorthAmerican Caucasians, 34% of African-Americans, and 55% ofAsian-Americans (Baur M P et al., Genet Epidemiol, 6:15-20 (1989)). Theidentification of the CD19 or CD20 epitope is performed by theevaluation of the amino acid sequence of CD19 or CD20 peptide motifsthat are likely to bind to HLA-A2.1.

Dendritic cells (DCs) have been used in the present experiments as anAPC because of their unique capacity to activate naïve T cells andinitiate primary antigen-specific T cell responses (Steinman A M, AnnuRev Immunol, 9:271-296 (1991); Porgador A et al., J Exp Med, 182:255-260(1995); Zitvogel L et al., J Exp Med, 183:87-97 (1996)). Presentation ofantigens by DCs may be especially important to inducing heightenedimmune responses to self-antigens since many immunization protocolstargeting self-antigens use whole cells and often result in theinduction of low-affinity CTL responses (Brossart P et al., J Exp Med,183:2449-2458 (1996); Houbiers J G et al., Eur J Immunol, 23:2072-2077(1993)). In this example, mature dendritic cells are used to presentexogeneous peptide sufficiently to T cells to evoke the peptide-specificCTLs. Optimal maturation factors for DCs are also examined.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

All references disclosed herein, including those cited hereafter, arespecifically incorporated herein by reference thereto.

While preferred embodiments have been illustrated and described, itshould be understood that changes and modifications can be made thereinin accordance with ordinary skill in the art without departing from theinvention in its broader aspects as defined in the following claims.

1. An isolated leukemic antigen consisting of the peptide KLMSPKLYV (SEQID NO: 4) or a variant thereof having one or two conservative ornon-conservative amino acid substitutions that is capable of stimulatinga cytotoxic T-lymphocyte reaction against a cell expressing CD19.
 2. Theisolated leukemic antigen of claim 1 consisting of the peptide KLMSPKLYV(SEQ ID NO: 4).
 3. A pharmaceutical composition comprising the isolatedleukemic antigen of claim 1 and a pharmaceutically acceptable carrier.4. The pharmaceutical composition of claim 3 further comprising one ormore co-immunostimulatory molecules.
 5. A method for stimulating animmune effector cell response comprising contacting the isolatedleukemic antigen of claim 1 with an immune effector cell, therebystimulating the immune effector cell to respond against the isolatedleukemic antigen.
 6. The method of claim 5, wherein the immune effectorcell is a naïve T-lymphocyte or a memory T-lymphocyte.
 7. The method ofclaim 5 further comprising contacting the isolated leukemic antigen withan antigen presenting cell wherein the antigen presenting cell contactsthe isolated leukemic antigen with the immune effector cell.
 8. Themethod of claim 7, wherein the antigen presenting cell is a dendriticcell or a T2 cell.
 9. The method of claim 7, wherein the antigenpresenting cell contacts the immune effector cell in vivo or in vitro.10. The method of claim 5, wherein contacting the isolated leukemicantigen with the immune effector cell occurs in the presence of animmunostimulatory molecule.